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Posts Tagged ‘hyperglycemia’


Autocrine selection of GLP-1 binding site

Larry H. Bernstein, MD, FCAP, Curator

LPBI

Update 12/15/2015

TSRI Team Finds Unique Anti-Diabetes Compound

Scientists from The Scripps Research Institute (TSRI) have deployed a powerful new drug discovery technique to identify an anti-diabetes compound with a novel mechanism of action

http://www.technologynetworks.com/HTS/news.aspx?ID=186055

The finding may lead to a new type of diabetes treatment. Just as importantly, it demonstrates the potential of the new technique, which enables researchers to quickly find drug candidates that activate cellular receptors in desired ways.

“In principle, we can apply this technique to hundreds of other receptors like the one we targeted in this study to find disease treatments that are more potent and have fewer side effects than existing therapies. It has been a very productive cross-campus collaboration, so we’re hoping to build on its success as we continue to collaborate on interrogating potential therapeutic targets,” said Patricia H. McDonald, an assistant professor at TSRI’s Jupiter, Florida campus and a senior investigator of the study.

McDonald’s laboratory collaborated on the study with the laboratory of Richard A. Lerner, the Lita Annenberg Hazen Professor of Immunochemistry at TSRI’s La Jolla campus, and with other TSRI groups. Lerner has pioneered techniques for generating and screening large libraries of antibodies or proteins to find new therapies.

In Search of a Better Activator

Three years ago, Lerner and colleagues devised a technique called autocrine selection, which enables scientists to screen very large libraries of molecules to find those that not only bind a given cellular receptor but also activate it to bring about a desired therapeutic effect. Since then, the Lerner laboratory and collaborating scientists have used the technique to find new molecules that block cold virus infection, boost red blood cell production and kill cancer cells, among other effects.

For the new study, Lerner and his laboratory used the technique to target a receptor linked to type 2 diabetes, a life-shortening disease estimated to affect 30 million people in the US alone.

The GLP-1 receptor, as it is known, is expressed by insulin-producing “beta cells” in the pancreas. Several drugs that activate this receptor—drugs called GLP-1 receptor agonists—are already approved for treating type 2 diabetes. In this case, the TSRI team’s aim was to find a molecule that activates the GLP-1 receptor in a unique way.

The GLP-1 receptor belongs to a large class of receptors known as G protein-coupled receptors (GPCRs). Scientists recently have come to understand that when a molecule activates a GPCR, it doesn’t necessarily trigger a single chain of biochemical signals within the cell. In fact, most GPCR agonists trigger signals via multiple distinct pathways—one being via a so-called G protein and another via a protein known as beta-arrestin. In some cases, a “biased agonist” that principally activates just one of these pathways would work better than one that activates both.

In this case, Lerner and his laboratory teamed up with McDonald, an expert on GPCRs and metabolic disease, to find a molecule that would preferentially activate the GLP-1 receptor’s G protein pathway.

To start, researchers in Lerner’s laboratory, including Hongkai Zhang, a senior staff scientist and co-first author of the study, generated a library of candidate molecules—based on a known GLP-1 receptor agonist, Exendin-4, a small protein (peptide) originally found in the venom of Gila monster lizards; a synthetic version of this protein is now used as a type 2 diabetes medication. Zhang created about one million new peptides by randomly varying one end of Exendin-4—the end that normally activates the G protein and beta arrestin pathways.

“The idea was that at least one of these many variants would induce a change in the shape of the GLP-1 receptor that would activate the G-protein pathway without activating the beta arrestin pathway,” Zhang said.

Using the autocrine selection system, Zhang and colleagues rapidly screened these variant peptides and eventually isolated one, P5, that potently and selectively activated the GLP-1 receptor’s G-protein pathway. An initial test in healthy mice showed that P5 worked well at boosting glucose tolerance—at about one-hundredth the dose of Exendin-4 needed for the same effect.

Protein expert Philip E. Dawson, an associate professor at TSRI’s La Jolla campus, synthesized sufficient quantities of P5, and McDonald and her laboratory performed more advanced tests in cultured cells and in mice.

A Different Mechanism

Exendin-4 and and other GLP-1 receptor agonists work in part by strongly stimulating pancreatic beta cells to produce more insulin—which signals muscle and fat cells to draw glucose from the blood, thus lowering blood glucose levels.

McDonald and her team found that although P5 equals or outperforms Exendin-4 in standard mouse models of diabetes, it stimulates insulin production only weakly.

“We didn’t expect that, but in fact, it was a nice finding because less reliance on stimulating insulin could mean less stress on the beta cells,” said Emmanuel Sturchler, staff scientist in the McDonald laboratory and co-first author of the study.

Investigating further, the team found that while the peptide doesn’t make mice fatter or heavier, it triggers the growth of new fat cells. In typical obesity-related diabetes, fat cells grow larger, not more numerous, and as they grow larger, they lose their ability to respond to insulin (insulin resistance). The proliferation of fat cells with P5 was accompanied by signs of increased insulin sensitivity in those cells, suggesting that the peptide works in part by alleviating insulin resistance.

Exendin-4 induces a feeling of satiety, causing mice (and people) to modestly lower food intake and thus lose weight. But the researchers found that P5 lacks this mechanism and appears to have no effect on appetite or weight.

“P5’s mechanisms of action turned out to be quite different from Exendin-4’s, and we think that this finding could lead to new therapeutics,” Sturchler said.

The team will now look for opportunities to develop P5 into a new diabetes drug. The researchers also see this as the first of many discoveries of GPCR-targeting compounds with unique and potentially valuable properties—as well as discoveries in basic GPCR biology.

 

New screening tech at Scripps spotlights diabetes drug candidates

Wednesday, December 9, 2015 | By John Carrol

 

The Scripps Research Institute has used a new drug screening platform to identify a drug which researchers believe has strong potential for treating diabetes.

Working with a technique dubbed autocrine selection, investigators are able to screen molecules in search of targets that can bind to and activate cellular receptors in order to achieve a sought-after drug effect.

In this latest study, published in Nature Communications, the Scripps team went after the GLP-1 receptor, which is already the target of a number of GLP-1 agonists. Scripps, though, wanted to activate the GLP-1 receptor’s G protein pathway.

Hongkai Zhang focused on the GLP-1 agonist Extendin-4, whipping up a million peptides that could alter the end of the protein that activates the G protein and beta arrestin pathways.

“The idea was that at least one of these many variants would induce a change in the shape of the GLP-1 receptor that would activate the G-protein pathway without activating the beta arrestin pathway,” Zhang said.

They then identified the one in a million that improved glucose tolerance at a radically reduced dose of Extendin-4, testing it on mice.

“P5’s mechanisms of action turned out to be quite different from Exendin-4’s, and we think that this finding could lead to new therapeutics,” said Emmanuel Sturchler, a staff scientist in the McDonald laboratory and co-first author of the study.

https://www.scripps.edu/news/press/2015/20151207lerner-mcdonald.html

Scientists from The Scripps Research Institute (TSRI) have deployed a powerful new drug discovery technique to identify an anti-diabetes compound with a novel mechanism of action.

The finding, which appeared online ahead of print in Nature Communications, may lead to a new type of diabetes treatment. Just as importantly, it demonstrates the potential of the new technique, which enables researchers to quickly find drug candidates that activate cellular receptors in desired ways.

“In principle, we can apply this technique to hundreds of other receptors like the one we targeted in this study to find disease treatments that are more potent and have fewer side effects than existing therapies. It has been a very productive cross-campus collaboration, so we’re hoping to build on its success as we continue to collaborate on interrogating potential therapeutic targets,” said Patricia H. McDonald, an assistant professor at TSRI’s Jupiter, Florida campus and a senior investigator of the study.

 

‘Fingerprints’ for Major Drug Development Targets

For the first time, scientists from the Florida campus of The Scripps Research Institute (TSRI) have created detailed “fingerprints” of a class of surface receptors that have proven highly useful for drug development.

http://www.technologynetworks.com/HTS/news.aspx?ID=185860

These detailed “fingerprints” show the surprising complexity of how these receptors activate their binding partners to produce a wide range of signaling actions.

The study focuses on interactions of G protein-coupled receptors (GPCRs) with their signaling mediators known as G proteins. GPCRs—currently accounting for about 40 percent of all prescription pharmaceuticals on the market—play key roles in many physiological functions because they transmit signals from outside the cell to the interior. When an outside substance binds to a GPCR, it activates a G protein inside the cell to release components and create a specific cellular response.

“Until now, it was generally believed that GPCRs are very selective, activating only a few G proteins they were designed to work with,” said TSRI Associate Professor Kirill Martemyanov, who led the study. “It turns out the reality is much more complex.”

Ikuo Masuho, a senior research associate in the Martemyanov lab, added, “Our imaging technology opens a unique avenue of developing drugs that would precisely control complex GPCR-G protein coupling, maximizing therapeutic potency by activating G proteins that contribute to therapeutic efficacy while inhibiting other G proteins that cause adverse side effects.”

The study found that individual GPCRs engage multiple G proteins with varying efficacy and rates, much like a dance where the most desirable partner, the GPCR, is surrounded by 14 suitors all vying for attention. The results, as in any dance, depend on which G proteins bind to the receptor—and for how long. The same receptor changes G protein partners—and the signaling outcome—depending on the action of the signal received from outside of the cell.

This finding was made possible by novel imaging technology used by the Martemyanov lab to monitor G protein activation in live cells. Using a pair of light-emitting proteins, one attached to the G protein, the other attached to what’s known as a reporter molecule, Martemyanov and his colleagues were able to measure simultaneously both the signal and activation rates of most G proteins present in the body.

“Our approach looks at 14 different types of G proteins at once—and we only have 16 in our bodies,” he said. “This is as close as it can get to what is actually happening in real time.”

In the accompanying commentary in Science Signaling, Alan Smrcka, a professor at University of Rochester Medical School and a prominent GPCR researcher, wrote, “[The findings] suggest the power of the GPCR fingerprinting approach, in that it could predict the G protein coupling specificity of a GPCR in a native system, which was previously undetected by conventional analysis. This could be very helpful for identifying previously unappreciated signaling pathways downstream of individual GPCRs that could be useful therapeutically or identified as potential side effects of GPCRs.”

 

Long-Acting Glucagon-Like Peptide 1 Receptor Agonists  

A review of their efficacy and tolerability

Alan J. Garber, MD, PHD

Diabetes Care May 2011; 34(Supplement 2): S279-S284    http://dx.doi.org/10.2337/dc11-s231

Targeting the incretin system has become an important therapeutic approach for treating type 2 diabetes. Two drug classes have been developed: glucagon-like peptide (GLP)-1 receptor agonists and dipeptidyl peptidase 4 (DPP-4) inhibitors. Clinical data have revealed that these therapies improve glycemic control while reducing body weight (GLP-1 receptor agonists, specifically) and systolic blood pressure (SBP) in patients with type 2 diabetes. Furthermore, incidence of hypoglycemia is relatively low with these treatments (except when used in combination with a sulfonylurea) because of their glucose-dependent mechanism of action. There are currently two GLP-1 receptor agonists available (exenatide and liraglutide), with several more currently being developed. This review considers the efficacy and safety of both the short- and long-acting GLP-1 receptor agonists. Head-to-head clinical trial data suggest that long-acting GLP-1 receptor agonists produce superior glycemic control when compared with their short-acting counterparts. Furthermore, these long-acting GLP-1 receptor agonists were generally well tolerated, with transient nausea being the most frequently reported adverse effect.

Careful consideration should be given to the selection of therapies for managing type 2 diabetes. In particular, antidiabetic agents that offer improved glycemic control without increasing cardiovascular risk factors or rates of hypoglycemia are warranted. At present, many available treatments for type 2 diabetes fail to maintain glycemic control in the longer term because of gradual disease progression as β-cell function declines. Where sulfonylureas or thiazolidinediones (common oral antidiabetic drugs) are used, the risk of hypoglycemia and weight gain can increase (1,2). The development of new therapies for the treatment of type 2 diabetes that, in addition to maintaining glycemic control, could reduce body weight and hypoglycemia risk (3,4), may help with patient management. Indeed, guidelines have been developed that support the consensus that blood pressure, weight reduction, and avoidance of hypoglycemic events should be targeted in type 2 diabetes management alongside glycemic targets. For example, the American Diabetes Association (ADA) defines multiple goals of therapy that include A1C <7.0% and SBP <130 mmHg and no weight gain (or, in the case of obese subjects, weight loss) (5). In particular, incretin-based therapies (GLP-1 receptor agonists, specifically) can help meet these new targets by offering weight reduction, blood pressure reduction, and reduced hypoglycemia in addition to glycemic control.

WHAT IS GLP-1?

The incretin effect, responsible for 50–70% of total insulin secretion after oral glucose administration, is defined as the difference in insulin secretory response from an oral glucose load compared with intravenous glucose administration (6) (Supplementary Fig. 1).

There are two naturally occurring incretin hormones that play a role in the maintenance of glycemic control: glucose-dependent insulinotropic polypeptide and GLP-1, both of which have a short half-life because of their rapid inactivation by DPP-4 (7). In patients with type 2 diabetes, the incretin effect is reduced or, in some cases, absent (8). In particular, the insulinoptropic action of glucose-dependent insulinotropic polypeptide is lost in patients with type 2 diabetes. However, it has been shown that, after administration of pharmacological levels of GLP-1, the insulin secretory function can be restored in this population (9), and thus GLP-1 has become an important target for research into new therapies for type 2 diabetes.

GLP-1 has multiple physiological effects that make it an attractive candidate for type 2 diabetes therapy. It increases insulin secretion while inhibiting glucagon release, but only when glucose levels are elevated (6,10), thus offering the potential to lower plasma glucose while reducing the likelihood of hypoglycemia. Furthermore, gastric emptying is delayed (10) and food intake is decreased after GLP-1 administration. Indeed, in a 6-week study investigating continuous GLP-1 infusion, patients with type 2 diabetes achieved a significant weight loss of 1.9 kg and a reduction in appetite from baseline compared with patients receiving placebo, where there was no significant change in weight or appetite (11). Preclinical studies reveal other potential benefits of GLP-1 receptor agonist treatment in individuals with type 2 diabetes, which include the promotion of β-cell proliferation (12) and reduced β-cell apoptosis (13). These preclinical results indicate that GLP-1 could be beneficial in treating patients with type 2 diabetes. However, because native GLP-1 is rapidly inactivated and degraded by the enzyme DPP-4 and has a very short half-life of 1.5 min (14), to achieve the clinical potential for native GLP-1, patients would require 24-h administration of native GLP-1 (15). Because this is impractical as a therapeutic option for type 2 diabetes, it was necessary to develop longer-acting derivatives of GLP-1.

DEVELOPMENT OF DPP-4–RESISTANT GLP-1 RECEPTOR AGONISTS

Two classes of incretin-based therapy have been developed to overcome the clinical limitations of native GLP-1: GLP-1 receptor agonists (e.g., liraglutide and exenatide), which exhibit increased resistance to DPP-4 degradation and thus provide pharmacological levels of GLP-1, and DPP-4 inhibitors (e.g., sitagliptin, vildagliptin, saxagliptin), which reduce endogenous GLP-1 degradation, thereby providing physiological levels of GLP-1. In this review, we focus on the GLP-1 receptor agonist class of incretin-based therapies. The efficacy and tolerability of the DPP-4 inhibitors have been reviewed elsewhere (16). Two GLP-1 receptor agonists are licensed at present in Europe, the U.S., and Japan: exenatide (Byetta, Eli Lilly) (17) and liraglutide (Victoza, Novo Nordisk) (18). For the purposes of this review, we refer to “short-acting” GLP-1 receptor agonists as those agents having duration of action of <24 h and “long-acting” as those agents with duration of action >24 h (Table 1).

….. more        http://care.diabetesjournals.org/content/34/Supplement_2/S279.full.pdf+html

 

Autocrine selection of a GLP-1R G-protein biased agonist with potent antidiabetic effects

Hongkai ZhangEmmanuel SturchlerJiang ZhuAinhoa NietoPhilip A. Cistrone,…., Patricia H. McDonald & Richard A. Lerner
Nature Communications Dec 2015; 6(8918)
       
     http://dx.doi.org:/10.1038/ncomms9918

Glucagon-like peptide-1 (GLP-1) receptor (GLP-1R) agonists have emerged as treatment options for type 2 diabetes mellitus (T2DM). GLP-1R signals through G-protein-dependent, and G-protein-independent pathways by engaging the scaffold protein β-arrestin; preferential signalling of ligands through one or the other of these branches is known as ‘ligand bias’. Here we report the discovery of the potent and selective GLP-1R G-protein-biased agonist, P5. We identified P5 in a high-throughput autocrine-based screening of large combinatorial peptide libraries, and show that P5 promotes G-protein signalling comparable to GLP-1 and Exendin-4, but exhibited a significantly reduced β-arrestin response. Preclinical studies using different mouse models of T2DM demonstrate that P5 is a weak insulin secretagogue. Nevertheless, chronic treatment of diabetic mice with P5 increased adipogenesis, reduced adipose tissue inflammation as well as hepatic steatosis and was more effective at correcting hyperglycemia and lowering hemoglobin A1clevels than Exendin-4, suggesting that GLP-1R G-protein-biased agonists may provide a novel therapeutic approach to T2DM.

Figure 1: Autocrine-based system for selection of agonists from large combinatorial peptide libraries

Autocrine-based system for selection of agonists from large combinatorial peptide libraries.

(a) Schematic representation of the peptide libraries. (b) Schematic representation of the membrane-tethered Exendin-4 (top) and FACS analysis of mCherry and GFP expression 2 days after transduction of HEK293-GLP-1R-GFP cells with the membrane-tethered Exendin-4 displaying different linker size (bottom). (c) Schematic representation of the autocrine-based selection of combinatorial peptide library. The lentivirus peptide libraries are preparred from lentiviral plasmids (step 1). The CRE-responsive GLP-1R reporter cell line is transduced with lentiviral library (step 2). GFP expressing cells are sorted (step 3) and peptide-encoding genes are amplified from genomic DNA of sorted cells to make the library for the next selection round (step 4). After iterative rounds of selection, enriched peptide sequences are analysed by deep sequencing (step 5). (d) Enrichment of GFP positive cells during three rounds of FACS selection. (e) N termini sequences of top 13 peptides (frequency>1.0% representation).

 

Type 2 diabetes mellitus (T2DM) is a complex metabolic disorder characterized by hyperglycaemia arising from a combination of insufficient insulin secretion together with the development of insulin resistance. The incretin, glucagon-like peptide-1 (GLP-1) is an endogenous peptide hormone secreted from intestinal endocrine cells in response to food intake1. GLP-1 lowers postprandial glucose excursion by potentiating glucose-stimulated insulin secretion from pancreatic β-cells and has also recently been shown to promote β-cell survival in rodents2. In addition, GLP-1 exerts extra-pancreatic actions such as promoting gastric emptying, weight loss and increasing insulin sensitivity in peripheral tissues3. Hence, incretin-based therapies represent a strategy for the treatment of T2DM.

GLP-1 exerts its action through the GLP-1 receptor (GLP-1R)4 expressed in the pancreas, other peripheral tissues, and the central nervous system. Activation of GLP-1R triggers Gαs-protein coupling leading to an elevation of cyclic AMP (cAMP), modulates intracellular calcium concentration5 and induces β-arrestin recruitment6, 7. Historically, β-arrestins were believed to serve an exclusive role in G-protein coupled receptor (GPCR) desensitization8. However, it has since been shown that β-arrestins can also function to activate signalling cascades9, 10. In this regard, in the pancreatic β-cell, elevation of both cAMP and cytosolic Ca2+ and β-arrestin signalling downstream of GLP-1R activation are critical events in promoting glucose-dependent insulin secretion.

Recently, the concept of ‘functional selectivity’ or ‘ligand bias’ has emerged whereby ligand binding promotes engagement of only a particular subset of the full GPCR signalling repertoire to the exclusion of others11. A better understanding of GLP-1R pleiotropic signalling and the underlying physiological consequences might provide new avenues for the development of drugs with novel modes of action that have the potential to provide greater therapeutic value while possibly avoiding unwanted side effects12, 13. Therefore we developed an autocrine-based system, to screen large and diverse, combinatorial peptide libraries containing up to 100 million different members with the aim of identifying potent, selective, G-protein-biased GLP-1R agonists. We identified one such ligand, designated P5 and have characterized its in vitro pharmacological phenotype, and explored its therapeutic potential.

P5 is a selective and potent G-protein-biased GLP-1R agonist

To assess potential signalling bias, the active peptides were further characterized in vitro using distinct assays that monitor receptor proximal signals. Cell-based assays for Gαs-protein (cAMP production), Gαq-protein (intracellular Ca2+ mobilization) and β-arrestin (1 and 2) signalling were used to determine the potency (EC50; effector concentration for half-maximum response) and maximal efficacy (Emax (%)) of peptides relative to the reference ligand Ex4 (Table 1). Peptides P1, P2, P5 and P10 all stimulated cAMP production. However, only P5 functioned as a full agonist (Emax=100%) displaying sub-nanomolar potency at both the human (hGLP-1R) and mouse receptor (mGLP-1R) (Fig. 2a,b; Table 1). The P5 EC50 was similar to the endogenous ligand GLP-1 but was slightly right shifted when compared with the reference peptide Ex4 (Fig. 2a,b; Table 1). Importantly, P5-induced cAMP production was inhibited by the selective GLP-1R antagonist Ex 9–39 in a concentration-dependent manner (Supplementary Fig. 1a,b). In addition, P5-induced cAMP production was negligible in HEK293 cells expressing the human glucagon receptor (Supplementary Fig. 1c). These data suggest that P5 selectively interacts with the GLP-1R.

 

In line with previous reports43, 44, 45 our data support the notion that non β-cell actions of GLP-1 agonists can improve glycaemic control. Importantly, GLP-1R is expressed in adipose tissue, in both the stromal vascular and the adipocyte fraction and its expression level has been found to correlate with the degree of insulin resistance46. In addition, the GLP-1 peptide has been reported to regulate adipogenesis in vitro47, 48. Given that P5, a G-protein-biased agonist with a severely blunted β-arrestin response has less propensity to induce GLP-1R desensitization, sustained activation of the receptor in adipose tissue may lead to the changes we observed in eWAT. Consistent with this notion, increased expression of adipogenic genes and a decrease in resistin expression was reported in β-arrestin 1 knockout mice49. Nevertheless, considering the multitude of metabolic pathways regulated by β-arrestin, further studies are warranted to determine the role of β-arrestin signalling downstream of GLP-1R activation in adipogenesis. Additionally, we found that chronic treatment with P5 increased circulating level of GIP to a greater extent than Ex4. Several studies demonstrated that GIP acts as an insulin sensitizer in adipocytes and disruption of the GIP/GIP-R axis has been reported in insulin-resistant states such as obesity50, 51. Interestingly, PPARγ activation was shown to increase GIP-R levels during adipocyte differentiation52. Thus, by increasing GIP and PPARγ levels, P5 chronic treatment may restore GIP/GIP-R signalling in adipocytes. Furthermore, previous studies have demonstrated that the simultaneous activation of the GLP-1R and the GIP-R results in enhanced glycaemic control, and lower HbA1c levels in human and rat, when compared with GLP-1R alone53, suggesting a GIP and GLP-1 synergism. Thus, the superior glycaemic control observed with the G-protein-biased agonist may result from P5-induced increases in GIP level and concomitant receptor activation. In addition, the GLP-1R can form homodimers as well as ligand-induced heterodimers with the GIP-R54. It is conceivable, that P5 may promote the formation of new and pharmacologically distinct homo/heterodimers displaying different signalling capacity. However, further studies are required to delineate more precisely the molecular and cellular mechanisms and the consequences of P5-induced increase in GIP levels.

In summary, high-throughput autocrine-based functional screening of combinatorial peptide libraries enabled the discovery of a high potency G-protein-biased GLP-1R agonist demonstrating new pharmacological virtues. In a series of translational preclinical studies we demonstrate that P5 is a weak insulin secretagogue yet displays superior antidiabetic effect (Fig. 7). Thus, GLP-1R G-protein-biased ligands may offer new and unappreciated advantages in the context of chronic treatment such as promoting adipocyte hyperplasia, restoring insulin responsiveness and long-term glycaemic control while preserving pancreatic β-cell function by minimizing the insulin secretory burden.

 

Figure 7: Schematic depicting the identification and characterization of a novel GLP-1R-biased agonist.

Schematic depicting the identification and characterization of a novel GLP-1R-biased agonist.

Using an autocrine-based system coupled to FACS, we screened large, diverse, combinatorial peptide libraries and identified P5, a potent and selective G-protein-biased GLP-1R agonist. P5 displayed a decreased insulinotropic effect, yet significantly improved glucose tolerance and insulin responsiveness by promoting white adipocyte tissue hyperplasia.

 

Exendin-4 Is a High Potency Agonist and Truncated Exendin-(9-39)- amide an Antagonist at the Glucagon-like Peptide 1-(7-36)-amide Receptor of Insulin-secreting ,&Cells*

Riidiger Goke, Hans-Christoph Fehmann, Thomas LinnS, Harald Schmidt, Michael Krause9, John EngT, and Burkhard GokeII
J Biol Chem  Sept 1993;268(26):19650-19655      http://www.jbc.org/content/268/26/19650.full.pdf

Exendin-4 purified from Heloderma suspecturn venom shows structural relationship to the important incretin hormone glucagon-like peptide 1-(7-36)- amide (GLP-1). We demonstrate that exendin-4 and truncated exendin-(9-39)-amide specifically interact with the GLP-1 receptor on insulinoma-derived cells and on lung membranes. Exendin-4 displaced “‘IGLP- 1, and unlabeled GLP- 1 displaced lZ6I-exendin-4 from the binding site at rat insulinoma-derived RINmSF cells. Exendin-4 had, like GLP-1, a pronounced effect on intracellular CAMP generation, which was reduced by exendin-(9-39)-amide. When combined, GLP-1 and exendin-4 showed additive action on CAMP. They each competed with the radiolabeled version of the other peptide in cross-linking experiments. The apparent molecular mass of the respective ligand-binding protein complex was 63,000 Da. Exendin-(9-39)-amide abolished the cross-linking of both peptides. Exendin-4, like GLP-1, stimulated dose dependently the glucose-induced insulin wcretion in isolated rat islets, and, in mouse insulinoma TC-1 cells, both peptides stimulated the proinsulin gene expression at the level of transcription. Exendin- (9-39)-amide reduced these effects. In conclusion, exendin-4 is an agonist and exendin-(9-39)-amide is a specific GLP- 1 receptor antagonist.

 

Glucagon-like peptide-1 receptor agonists for the treatment of type 2 diabetes mellitus

Kathleen Dungan, MDAnthony DeSantis, MD
http://www.uptodate.com/contents/glucagon-like-peptide-1-receptor-agonists-for-the-treatment-of-type-2-diabetes-mellitus

Despite advances in options for the treatment of diabetes, optimal glycemic control is often not achieved. Hypoglycemia and weight gain associated with many antidiabetic medications may interfere with the implementation and long-term application of “intensive” therapies [1]. Current treatments have centered on increasing insulin availability (either through direct insulin administration or through agents that promote insulin secretion), improving sensitivity to insulin, delaying the delivery and absorption of carbohydrate from the gastrointestinal tract, or increasing urinary glucose excretion.

Glucagon-like peptide-1 (GLP-1)-based therapies (eg, GLP-1 receptor agonists, dipeptidyl peptidase 4 [DPP-4] inhibitors) affect glucose control through several mechanisms, including enhancement of glucose-dependent insulin secretion, slowed gastric emptying, and reduction of postprandial glucagon and of food intake (table 1). These agents do not usually cause hypoglycemia in the absence of therapies that otherwise cause hypoglycemia.

This topic will review the mechanism of action and therapeutic utility of GLP-1 receptor agonists for the treatment of type 2 diabetes mellitus. DPP-4 inhibitors are discussed separately. A general discussion of the initial management of blood glucose and the management of persistent hyperglycemia in adults with type 2 diabetes is also presented separately. (See “Dipeptidyl peptidase 4 (DPP-4) inhibitors for the treatment of type 2 diabetes mellitus”.)

GLUCAGON-LIKE PEPTIDE-1

Glucose homeostasis is dependent upon a complex interplay of multiple hormones: insulin and amylin, produced by pancreatic beta cells; glucagon, produced by pancreatic alpha cells; and gastrointestinal peptides, including glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP; gastric inhibitory polypeptide) (figure 1). Abnormal regulation of these substances may contribute to the clinical presentation of diabetes. The role of GLP-1 in glucose homeostasis is illustrative of the incretin effect, in which oral glucose has a greater stimulatory effect on insulin secretion than intravenous glucose [2]. This effect is mediated by several gastrointestinal peptides, particularly GLP-1, that are released in the setting of a meal and stimulate insulin synthesis and insulin secretion, which does not occur when carbohydrate is administered intravenously.

GLP-1 is produced from the proglucagon gene in L-cells of the small intestine and is secreted in response to nutrients (figure 1) [3]. GLP-1 binds to a specific GLP-1 receptor, which is expressed in various tissues including pancreatic beta cells, pancreatic ducts, gastric mucosa, kidney, lung, heart, skin, immune cells, and the hypothalamus [2,4]. GLP-1 exerts its main effect by stimulating glucose-dependent insulin release from the pancreatic islets [2]. It has also been shown to slow gastric emptying [5], inhibit inappropriate post-meal glucagon release [3,6], and reduce food intake (table 1) [3]. Owing in part to the effects of GLP-1 on slowed gastric emptying and appetite centers in the brain, therapy with GLP-1 and its receptor agonists is associated with weight loss, even among patients without significant nausea and vomiting.

 

Exendin-4, a glucagon-like peptide-1 receptor agonist, reduces Alzheimer disease-associated tau hyperphosphorylation in the hippocampus of rats with type 2 diabetes.
Impaired insulin signaling pathway in the brain in type 2 diabetes (T2D) is a risk factor for Alzheimer disease (AD). Glucagon-like peptide-1 (GLP-1) and its receptor agonist are widely used for treatment of T2D. Here we studied whether the effects of exendin-4 (EX-4), a long-lasting GLP-1 receptor agonist, could reduce the risk of AD in T2D.  RESULTS: The levels of phosphorylated tau protein at site Ser199/202 and Thr217 level in the hippocampus of T2D rats were found to be raised notably and evidently decreased after EX-4 intervention. In addition, brain insulin signaling pathway was ameliorated after EX-4 treatment, and this result was reflected by a decreased activity of PI3K/AKT and an increased activity of GSK-3β in the hippocampus of T2D rats as well as a rise in PI3K/AKT activity and a decline in GSK-3β activity after 4 weeks intervention of EX-4. CONCLUSIONS: These results demonstrate that multiple days with EX-4 appears to prevent the hyperphosphorylation of AD-associated tau protein due to increased insulin signaling pathway in the brain. These findings support the potential use of GLP-1 for the prevention and treatment of AD in individuals with T2D.
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CaKMII Inhibition in Obese, Diabetic Mice leads to Lower Blood Glucose Levels

Reporter: Larry H Bernstein, MD, FCAP

This recent publication was reported in MedPage today. It is different than, but highly suggestive of our recent report about the Univesity of Iowa discovery of “Oxidized CaKMII inhibition” as a therapeutic target for atrial arrhythmia.

Oxidized Calcium Calmodulin Kinase and Atrial Fibrillation
Author: Larry H. Bernstein, MD, FCAP, and Curator: Aviva Lev-Ari, PhD, RN
https://pharmaceuticalintelligence.com/2013/10/26/oxidized-calcium-calmodulin-kinase-and-atrial-fibrillation/
This is a review of a recent work from the laboratory of Mark E. Anderson and associates at the University of Iowa.  We have covered the role of CaMKII in calcium signaling and myocardiocyte contraction, as well as signaling in smooth muscle, skeletal muscle, and nerve transmission.  There are tissue specific modus operandi, partly related to the ryanogen receptor, and also related to tissue specific isoenzymes of CaMKII.  There is much ground that has been traversed in exploring these mechanisms, most recently, the discoverey of hormone triggering by the release from vesicles at the nerve muscle junction, and much remains open to investigation.  The recently published work by Mark E. Anderson and associates in Mannheim and Heidelberg, Germany, clarifies the relationship between the oxidized form of CaMKII and the triggering of atrial fibrillation. The following studies show:
  • Ang II infusion increased the susceptibility of mice to AF induction by rapid right atrial pacing and established a framework for us to test the hypothesized role of ox-CaMKII in promoting AF. ox-CaMKII is critical for AF.
    • Established a critical role of ox-CaMKII in promoting AF
  • Ang II induced increases in ROS production seen in WT atria were absent in atria from MsrA TG mice suggesting that MsrA sensitive targets represent an important component of Ang II mediated atrial oxidation.
    • The protection from AF in MsrA TG mice appeared to be independent of pressor effects that are critical for the proarrhythmic actions.
  • These findings suggest that NADPH oxidase dependent ROS and elevated ox-CaMKII
    • drive Ang II -pacing-induced AF and that
  • targeted antioxidant therapy, by MsrA over-expression,
    • can reduce or prevent AF in Ang -II-infused mice.
Atrial myocytes from Ang II treated WT mice showed a significant (p<0.05) increase in spontaneous Ca2+ sparks compared to atrial myocytes from saline treated control mice
In contrast to findings in WT mice, the atrial myocytes isolated from Ang II treated MM-VV mice did not show an increase in Ca2+ sparks compared to saline treated MM-VV mice
These data to suggest that  in ox–the proarrhythmic effects of Ang II infusion depend upon an increaseCaMKII, sarcoplasmic reticulum Ca2+ leak and DADs.
Enhanced CaMKII-mediated phosphorylation of serine 2814 on RyR2
  • is associated with an increased susceptibility to acquired arrhythmias, including AF
Proarrhythmic actions of ox-CaMKII
  • require access to RyR2 serine 2814.
Mutant S2814A knock-in mice (lacking serine 2814) were highly resistant to Ang II mediated AF
AC3-I mice with transgenic myocardial expression of a CaMKII inhibitory peptide were also resistant to the proarrhythmic effects of Ang II infusion on pacing-induced AF
S2814A, AC3-I and WT mice, all developed similar BP increases and cardiac hypertrophy in response to Ang II, indicating that
  • these mice were not resistant to the hemodynamic effects of Ang II, but were nevertheless protected from AF.
selectively targeted antioxidant therapies could be effective in preventing or reducing AF
half of patients enrolled in the Mode Selection Trial (MOST) with sinus node dysfunction had a history of AF
Ang II and diabetes-induced CaMKII oxidation caused sinus node dysfunction by increased pacemaker cell death and fibrosis
 ox-CaMKII increases susceptibility for AF via increased diastolic sarcoplasmic reticulum Ca2+ release
clinical association between sinus node dysfunction and AF might have a mechanistic basis because
  • sinus node dysfunction and AF are downstream consequences of elevated ox-CaMKII.
We refer the reader to the following related articles published in pharmaceutical Intelligence:
  1. Contributions to cardiomyocyte interactions and signaling
    Author and Curator: Larry H Bernstein, MD, FCAP  and Curator: Aviva Lev-Ari, PhD, RN
    https://pharmaceuticalintelligence.com/2013/10/21/contributions-to-cardiomyocyte-interactions-and-signaling/
  2. Cardiac Contractility & Myocardium Performance: Therapeutic Implications for Ryanopathy (Calcium Release-related Contractile Dysfunction) and Catecholamine Responses
    Editor: Justin Pearlman, MD, PhD, FACC, Author and Curator: Larry H Bernstein, MD, FCAP, and Article Curator: Aviva Lev-Ari, PhD, RN
    https://pharmaceuticalintelligence.com/2013/08/28/cardiac-contractility-myocardium-performance-ventricular-arrhythmias-and-non-ischemic-heart-failure-therapeutic-implications-for-cardiomyocyte-ryanopathy-calcium-release-related-contractile/
  3. Part I. Identification of Biomarkers that are Related to the Actin Cytoskeleton
    Curator and Writer: Larry H Bernstein, MD, FCAP
    https://pharmaceuticalintelligence.com/2012/12/10/identification-of-biomarkers-that-are-related-to-the-actin-cytoskeleton/
  4. Part II: Role of Calcium, the Actin Skeleton, and Lipid Structures in Signaling and Cell Motility
    Larry H. Bernstein, MD, FCAP, Stephen Williams, PhD and Aviva Lev-Ari, PhD, RN
    https://pharmaceuticalintelligence.com/2013/08/26/role-of-calcium-the-actin-skeleton-and-lipid-structures-in-signaling-and-cell-motility/
  5. Part IV: The Centrality of Ca(2+) Signaling and Cytoskeleton Involving Calmodulin Kinases and Ryanodine Receptors in Cardiac Failure, Arterial Smooth Muscle, Post-ischemic Arrhythmia, Similarities and Differences, and Pharmaceutical Targets
    Larry H Bernstein, MD, FCAP, Justin Pearlman, MD, PhD, FACC and Aviva Lev-Ari, PhD, RN
    https://pharmaceuticalintelligence.com/2013/09/08/the-centrality-of-ca2-signaling-and-cytoskeleton-involving-calmodulin-kinases-and-ryanodine-receptors-in-cardiac-failure-arterial-smooth-muscle-post-ischemic-arrhythmia-similarities-and-differen/
  6. Part VI: Calcium Cycling (ATPase Pump) in Cardiac Gene Therapy: Inhalable Gene Therapy for Pulmonary Arterial Hypertension and Percutaneous Intra-coronary Artery Infusion for Heart Failure: Contributions by Roger J. Hajjar, MD
    Aviva Lev-Ari, PhD, RN
    https://pharmaceuticalintelligence.com/2013/08/01/calcium-molecule-in-cardiac-gene-therapy-inhalable-gene-therapy-for-pulmonary-arterial-hypertension-and-percutaneous-intra-coronary-artery-infusion-for-heart-failure-contributions-by-roger-j-hajjar/
  7. Part VII: Cardiac Contractility & Myocardium Performance: Ventricular Arrhythmias and Non-ischemic Heart Failure – Therapeutic Implications for Cardiomyocyte Ryanopathy (Calcium Release-related Contractile Dysfunction) and Catecholamine Responses
    Justin Pearlman, MD, PhD, FACC, Larry H Bernstein, MD, FCAP and Aviva Lev-Ari, PhD, RN
    https://pharmaceuticalintelligence.com/2013/08/28/cardiac-contractility-myocardium-performance-ventricular-arrhythmias-and-non-ischemic-heart-failure-therapeutic-implications-for-cardiomyocyte-ryanopathy-calcium-release-related-contractile/
  8. Part VIII: Disruption of Calcium Homeostasis: Cardiomyocytes and Vascular Smooth Muscle Cells: The Cardiac and Cardiovascular Calcium Signaling Mechanism
    Justin Pearlman, MD, PhD, FACC, Larry H Bernstein, MD, FCAP and Aviva Lev-Ari, PhD, RN
    https://pharmaceuticalintelligence.com/2013/09/12/disruption-of-calcium-homeostasis-cardiomyocytes-and-vascular-smooth-muscle-cells-the-cardiac-and-cardiovascular-calcium-signaling-mechanism/
  9. Part IX: Calcium-Channel Blockers, Calcium Release-related Contractile Dysfunction (Ryanopathy) and Calcium as Neurotransmitter Sensor
    Justin Pearlman, MD, PhD, FACC, Larry H Bernstein, MD, FCAP and Aviva Lev-Ari, PhD, RN
    https://pharmaceuticalintelligence.com/2013/09/16/calcium-channel-blocker-calcium-as-neurotransmitter-sensor-and-calcium-release-related-contractile-dysfunction-ryanopathy/
  10. Part X: Synaptotagmin functions as a Calcium Sensor: How Calcium Ions Regulate the fusion of vesicles with cell membranes during Neurotransmission
    Larry H Bernstein, MD, FCAP and Aviva Lev-Ari, PhD, RN
    https://pharmaceuticalintelligence.com/2013/09/10/synaptotagmin-functions-as-a-calcium-sensor-how-calcium-ions-regulate-the-fusion-of-vesicles-with-cell-membranes-during-neurotransmission/
  11. Genetic Analysis of Atrial Fibrillation
    Author and Curator: Larry H Bernstein, MD, FCAP ,  and Curator: Aviva-Lev Ari, PhD, RN
    https://pharmaceuticalintelligence.com/2013/10/27/genetic-analysis-of-atrial-fibrillation/
This article is a followup of the wonderful study of the effect of oxidation of a methionine residue in calcium dependent-calmodulin kinase Ox-CaMKII on stabilizing the atrial cardiomyocyte, giving protection from atrial fibrillation.  It is also not so distant from the work reviewed, mostly on the ventricular myocyte and the calcium signaling by initiation of the ryanodyne receptor (RyR2) in calcium sparks and the CaMKIId isoenzyme.

Diabetes: Mouse Studies Point to Kinase as Treatment Target

Published: Nov 24, 2013
By Kristina Fiore, Staff Writer, MedPage Today
Targeting a pathway that plays a major role in both hepatic glucose production and insulin sensitivity may eventually help treat type 2 diabetes, researchers reported.
In a series of experiments in mice, researchers found that inhibition of the kinase CaKMII — or even some of its downstream components — lowered blood glucose and insulin levels, Ira Tabas, MD, PhD, of Columbia University Medical Center in New York City, and colleagues reported online in Cell Metabolism.
The pathway is activated by glucagon signaling in the liver, and appears to have roles in both insulin resistance as well as hepatic glucose production in the liver.
In an earlier study, Tabas and colleagues showed that inhibiting the CaKMII pathway lowered hepatic glucose production by suppressing p38-mediated FoxO1 nuclear localization.
In the current study, they found CaKMII inhibition suppresses levels of the pseudo-kinase TRB3 to improve Akt-phosphorylation, thereby improving insulin sensitivity.
Thus this single pathway targets “two cardinal features of type 2 diabetes — hyperglycemia and defective insulin signaling,” the researchers wrote.
“When we realized we had one common pathway that was responsible for these two disparate processes that, in essence, comprises all of type 2 diabetes, we though it would be an ideal target for new drug therapy,” Tabas told MedPage Today.
Tabas and colleagues conducted several experiments to evaluate the CaKMII pathway.
In one experiment in obese mice, they found that no matter how CaKMII was knocked out, it led to lower blood glucose levels and lower fasting plasma insulin levels in response to a glucose challenge.
The improvements also occurred when they
  • knocked out downstream processes, including p38 and MAPK-activating protein kinase 2 (MK2).
“Thus liver p38 and MK2, like CaKMII, play an important role in the development of hyperglycemia and hyperinsulinemia in obese mice,” they wrote.
In further analyses, the researchers discovered deleting or inhibiting any of these three elements ultimately
  • improved insulin-induced Akt-phosphorylation in obese mice —
  • an important part of improving insulin sensitivity.
And unlike the effects on hepatic glucose production,
  • these changes didn’t occur through effects on FoxO1.
Instead, inhibiting the CaKMII pathway suppressed levels of the pseudo-kinase TRB3, which likely occurred because of
  • suppression of ATF4 — all of which led to an
  • increase in Akt-phosphorylation and insulin sensitivity.
Indeed, when mice were made to overexpress TRB3, the improvement in phosphorylation disappeared, “indicating that
  • the suppression of TRB3 by CaKMII deficiency is
  • causally important in the improvement in insulin signaling,
As a result, there “appear to be two separate CaKMII pathways”,
  1. one involved in CaKMII-p38-FoxO1 dependent hepatic glucose production, and
  2. the other involved in defective insulin-induced p-Akt,
The findings suggest the possibility of a drug that can target
  • both hyperglycemia and insulin resistance in type 2 diabetes
The authors have started developing such an agent. Although kinases can act very generally, Tabas said he and colleagues are working on
  • an allosteric version that will more specifically target MK2
  • by binding to a site that is unique to this enzyme.
He said this should help to avoid problems with drugs that targeted p38 but ultimately failed, with little efficacy and too many side effects.
The reason for this is now known at a very detailed level –
  • when you inhibit p38 by that mechanism, mainly by inhibiting MK2,
  • you avoid the adverse effects,
“When we realized all of this and had to make a choice [for further development], the obvious choice was MK2.”
  • CaKMII inhibitors are in development for heart failure and
  • MK2 inhibitors are being looked at as an alternative to p38 inhibitors for inflammatory diseases.
Tabas also said the drug may be valuable in treating prediabetes, since early data have suggested that
  • CaKMII is generally overactive in obese patients
  • who have not yet progressed to full blown diabetes, but is not overactive in lean people.
“One of the areas we’re most excited about in potential clinical use is in obese people before they get diabetic,” Tabas told MedPage Today. “There are hundreds of millions of people who are obese but not yet diabetic even though
  • they have the hallmarks that they’re going to get diabetes.”
This recent publication was reported in MedPage Today. [CaKMII overactivity in obesity]  Tabas noted that his group’s early human data “suggest that our pathway is turned on in prediabetes. If we can block that pathway before people get diabetes, it would even be better.”
The study was supported by the NIH, the American Heart Association, the German Center for Cardiovascular Research, the German Ministry of Education and Research, and the European Union.
Tabas and a co-author are among the founders of  Tabomedex Biosciences, which is developing MK2 inhibitors.
Primary source: Cell Metabolism
Source reference: Ozcan L, et al. “Activation of calcium/calmodulin-dependent protein kinase II in obesity mediates suppression of hepatic insulin signaling” Cell Metab 2013.

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Reporter and Curator: Dr. Sudipta Saha, Ph.D.

Antioxidant micronutrients, such as vitamins and carotenoids, exist in abundance in fruit and vegetables and have been known to contribute to the body’s defence against reactive oxygen species. Numerous epidemiological studies have demonstrated that a high dietary consumption of fruit and vegetables rich in carotenoids or with high serum carotenoid concentrations results in lower risks of certain cancers, diabetes and cardiovascular disease. These epidemiological studies have suggested that antioxidant carotenoids may have a protective effect against diabetes or cardiovascular disease. However, the consumption of carotenoids in pharmaceutical forms for the treatment or prevention of these chronic diseases cannot be recommended, because some large randomized controlled trials did not reveal any reduction in cardiovascular events or type 2 diabetes with b-carotene. High doses of carotenoids used in the supplementation studies could have a pro-oxidant effect. Therefore, it is favourable to intake carotenoids from foods through the combination of other nutrients such as vitamins, minerals or phytochemicals, not by supplements.

The metabolic syndrome is a clustering of metabolic abnormalities that increase the risk for diabetes and cardiovascular disease. Typically, it includes excess weight, hyperglycaemia, evaluated blood pressure, low concentration of HDL-cholesterol, and hypertriacylglycerolaemia. This syndrome is emerging as one of the major medical and public health problems in Japan, and persons with this syndrome have an increased risk of morbidity and mortality due to cardiovascular disease and diabetes. Recently, many studies have examined the associations of dietary patterns with the metabolic syndrome and shown that diets rich in fruit and vegetables have been inversely associated with the metabolic syndrome. These previous reports suggest that a high intake of fruit and vegetables may reduce the risk of the metabolic syndrome through the beneficial combination of antioxidants, fibre, minerals, and other phytochemicals. Some recent cross-sectional and case–control studies have shown the associations of serum antioxidant status with the metabolic syndrome. Ford et al. reported that low intake and/or low serum concentrations of vitamins and carotenoids were associated with the risk of the metabolic syndrome. Although very few data are available about the associations of antioxidant carotenoids with the metabolic syndrome, people who have the metabolic syndrome are more likely to have increased oxidative stress than people who do not have this syndrome.

In some recent studies, it has been reported that oxidative stress, which is an imbalance between pro-oxidants and antioxidants, occurs more frequently in metabolic syndrome subjects than in non-metabolic syndrome subjects. Oxidative stress may play a key role in the pathophysiology of diabetes and cardiovascular disease. On the other hand, smoking is a potent oxidative stress in man. This increment of oxidative stress induced by smoking may develop insulin resistance, and increased insulin resistance may result in the clustering of the metabolic abnormality. Therefore, antioxidants could have a beneficial effect on reducing the risk of these conditions in smokers. However, there is limited information about the interaction of serum antioxidant carotenoids and the metabolic syndrome with smoking habit. This study was aimed to investigate the interaction of serum carotenoid concentrations and the metabolic syndrome with smoking. The association of the concentrations of six serum carotenoids, i.e. lutein, lycopene, a-carotene, b-carotene, b-cryptoxanthin and zeaxanthin, with metabolic syndrome status stratified by smoking status was evaluated crosssectionally.

In this study, the associations of the serum carotenoids with the metabolic syndrome stratified by smoking habit were evaluated cross-sectionally. A total of 1073 subjects (357 male and 716 female) who had received health examinations in the town of Mikkabi, Shizuoka Prefecture, Japan, participated in the study. Inverse associations of serum carotenoids with the metabolic syndrome were more evident among current smokers than non-smokers. These results support that antioxidant carotenoids may have a protective effect against development of the metabolic syndrome, especially in current smokers who are exposed to a potent oxidative stress.

Source References:

http://www.ncbi.nlm.nih.gov/pubmed/18445303

http://www.ncbi.nlm.nih.gov/pubmed/19450371

http://www.ncbi.nlm.nih.gov/pubmed/21216053

http://www.ncbi.nlm.nih.gov/pubmed/19631019

http://www.ncbi.nlm.nih.gov/pubmed/12324189

http://www.ncbi.nlm.nih.gov/pubmed/18689373

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Special Considerations in Blood Lipoproteins, Viscosity, Assessment and Treatment


Special Considerations in Blood Lipoproteins, Viscosity, Assessment and Treatment

Author: Larry H. Bernstein, MD, FCAP

and

Curator: Aviva Lev-Ari, PhD, RN

This is the second of a two part discussion of viscosity, hemostasis, and vascular risk

This is Part II of a series on blood flow and shear stress effects on hemostasis and vascular disease.

See Part I on viscosity, triglycerides and LDL, and thrombotic risk.

 

Hemostatic Factors in Thrombophilia

Objectives.—To review the state of the art relating to elevated hemostatic factor levels as a potential risk factor for thrombosis, as reflected by the medical literature and the consensus opinion of recognized experts in the field, and to make recommendations for the use of specific measurements of hemostatic factor levels in the assessment of thrombotic risk in individual patients.

Data Sources.—Review of the medical literature, primarily from the last 10 years.

Data Extraction and Synthesis.—After an initial assessment of the literature, key points were identified. Experts were assigned to do an in-depth review of the literature and to prepare a summary of their findings and recommendations.

A draft manuscript was prepared and circulated to every participant in the College of American Pathologists Conference XXXVI: Diagnostic Issues in Thrombophilia prior to the conference. Each of the key points and associated recommendations was then presented for discussion at the conference. Recommendations were accepted if a consensus of the 27 experts attending the conference was reached. The results of the discussion were used to revise the manuscript into its final form.

Consensus was reached on 8 recommendations concerning the use of hemostatic factor levels in the assessment of thrombotic risk in individual patients.

The underlying premise for measuring elevated coagulation factor levels is that if the average level of the factor is increased in the patient long-term, then the patient may be at increased risk of thrombosis long-term. Both risk of thrombosis and certain factors increase with age (eg, fibrinogen, factor VII, factor VIII, factor IX, and von Willebrand factor). Are these effects linked or do we need age specific ranges? Do acquired effects like other diseases or medications affect factor levels, and do the same risk thresholds apply in these instances? How do we assure that the level we are measuring is a true indication of the patient’s average baseline level and not a transient change? Fibrinogen, factor VIII, and von Willebrand factor are all strong acute-phase reactants.

Risk of bleeding associated with coagulation factor levels increases with roughly log decreases in factor levels. Compared to normal (100%), 60% to 90% decreases in a coagulation factor may be associated with excess bleeding with major trauma, 95% to 98% decreases with minor trauma, and .99% decrease with spontaneous hemorrhage. In contrast, the difference between low risk and high risk for thrombosis may be separated by as little as 15% above normal.

It may be possible to define relative cutoffs for specific factors, for example, 50% above the mean level determined locally in healthy subjects for a certain factor. Before coagulation factor levels can be routinely used to assess individual risk, work must be done to better standardize and calibrate the assays used.

Detailed discussion of the rationale for each of these recommendations is presented in the article. This is an evolving area of research. While routine use of factor level measurements is not recommended, improvements in assay methodology and further clinical studies may change these recommendations in the future.

Chandler WL, Rodgers GM, Sprouse JT, Thompson AR.  Elevated Hemostatic Factor Levels as Potential Risk Factors for Thrombosis.  Arch Pathol Lab Med. 2002;126:1405–1414

Model System for Hemostatic Behavior

This study explores the behavior of a model system in response to perturbations in

  • tissue factor
  • thrombomodulin surface densities
  • tissue factor site dimensions
  • wall shear rate.

The classic time course is characterized by

  • initiation and
  • amplification of thrombin generation
  • the existence of threshold-like responses

This author defines a new parameter, the „effective prothrombotic zone‟,  and its dependence on model parameters. It was found that prothrombotic effects may extend significantly beyond the dimensions of the spatially discrete site of tissue factor expression in both axial and radial directions. Furthermore, he takes advantage of the finite element modeling approach to explore the behavior of systems containing multiple spatially distinct sites of TF expression in a physiologic model. The computational model is applied to assess individualized thrombotic risk from clinical data of plasma coagulation factor levels. He proposes a systems-based parameter with deep venous thrombosis using computational methods in combination with biochemical panels to predict hypercoagulability for high risk populations.

 

The Vascular Surface

The ‘resting’ endothelium synthesizes and presents a number of antithrombogenic molecules including

  • heparan sulfate proteoglycans
  • ecto-adenosine diphosphatase
  • prostacyclin
  • nitric oxide
  • thrombomodulin.

In response to various stimuli

  • inflammatory mediators
  • hypoxia
  • oxidative stress
  • fluid shear stress

the cell surface becomes ‘activated’ and serves to organize membrane-associated enzyme complexes of coagulation.

Fluid Phase Models of Coagulation

Leipold et al. developed a model of the tissue factor pathway as a design aid for the development of exogenous serine protease inhibitors. In contrast, Guo et al. focused on the reactions of the contact, or intrinsic pathway, to study parameters relevant to material-induced thrombosis, including procoagulant surface area.

Alternative approaches to modeling the coagulation cascade have been pursued including the use of stochastic activity networks to represent the intrinsic, extrinsic, and common pathways through fibrin formation and a kinetic Monte Carlo simulation of TF-initiated thrombin generation. Generally, fluid phase models of the kinetics of coagulation are both computationally and experimentally less complex. As such, the computational models are able to incorporate a large number of species and their reactions, and empirical data is often available for regression analysis and model validation. The range of complexity and motivations for these models is wide, and the models have been used to describe various phenomena including the ‘all-or-none’ threshold behavior of thrombin generation. However, the role of blood flow in coagulation is well recognized in promoting the delivery of substrates to the vessel wall and in regulating the thrombin response by removing activated clotting factors.

Flow Based Models of Coagulation

In 1990, Basmadjian presented a mathematical analysis of the effect of flow and mass transport on a single reactive event at the vessel wall and consequently laid the foundation for the first flow-based models of coagulation. It was proposed that for vessels greater than 0.1 mm in diameter, reactive events at the vessel wall could be adequately described by the assumption of a concentration boundary layer very close to the reactive surface, within which the majority of concentration changes took place. The height of the boundary layer and the mass transfer coefficient that described transport to and from the vessel wall were shown to stabilize on a time scale much shorter than the time scale over which concentration changes were empirically observed. Thus, the vascular space could be divided into two compartments, a boundary volume and a bulk volume, and furthermore, changes within the bulk phase could be considered negligible, thereby reducing the previously intractable problem to a pseudo-one compartment model described by a system of ordinary differential equations.

Basmadjian et al. subsequently published a limited model of six reactions, including two positive feedback reactions and two inhibitory reactions, of the common pathway of coagulation triggered by exogenous factor IXa under flow. As a consequence of the definition of the mass transfer coefficient, the kinetic parameters were dependent on the boundary layer height. Furthermore, the model did not explicitly account for intrinsic tenase or prothrombinase formation, but rather derived a rate expression for reaction in the presence of a cofactor. The major finding of the study was the predicted effect of increased mass transport to enhance thrombin generation by decreasing the induction time up to a critical mass transfer rate, beyond which transport significantly decreased peak thrombin levels thereby reducing overall thrombin production.

Kuharsky and Fogelson formulated a more comprehensive, pseudo-one compartment model of tissue factor-initiated coagulation under flow, which included the description of 59 distinct fluid- and surface-bound species. In contrast to the Baldwin-Basmadjian model, which defined a mass transfer coefficient as a rate of transport to the vessel surface, the Kuharsky-Fogelson model defined the mass transfer coefficient as a rate of transport into the boundary volume, thus eliminating the dependence of kinetic parameters on transport parameters. The computational study focused on the threshold response of thrombin generation to the availability of membrane binding sites. Additionally, the model suggested that adhered platelets may play a role in blocking the activity of the TF/ VIIa complex. Fogelson and Tania later expanded the model to include the protein C and TFPI pathways.

Modeling surface-associated reactions under flow uses finite element method (FEM), which is a technique for solving partial differential equations by dividing the vascular space into a finite number of discrete elements. Hall et al. used FEM to simulate factor X activation over a surface presenting TF in a parallel plate flow reactor. The steady state model was defined by the convection-diffusion equation and Michaelis-Menten reaction kinetics at the surface. The computational results were compared to experimental data for the generation of factor Xa by cultured rat vascular smooth muscle cells expressing TF.

Based on discrepancies between numerical and experimental studies, the catalytic activity of the TF/ VIIa complex may be shear-dependent. Towards the overall objective of developing an antithrombogenic biomaterial, Tummala and Hall studied the kinetics of factor Xa inhibition by surface-immobilized recombinant TFPI under unsteady flow conditions. Similarly, Byun et al. investigated the association and dissociation kinetics of ATIII inactivation of thrombin accelerated by surface-immobilized heparin under steady flow conditions. To date, finite element models that detail surface-bound reactions under flow have been restricted to no more than a single reaction catalyzed by a single surface-immobilized species.

 

Models of Coagulation Incorporating Spatial Parameter

Major findings include the roles of these specific coagulation pathways in the

  • initiation
  • amplification
  • termination phases of coagulation.

Coagulation near the activating surface was determined by TF/VIIa catalyzed factor Xa production, which was rapidly inhibited close to the wall. In contrast, factor IXa diffused farther from the surface, and thus factor Xa generation and clot formation away from the reactive wall was dependent on intrinsic tenase (IXa/ VIIIa) activity. Additionally, the concentration wave of thrombin propagated away from the activation zone at a rate which was dependent on the efficiency of inhibitory mechanisms.

Experimental and ‘virtual’ addition of plasma-phase thrombomodulin resulted in dose-dependent termination of thrombin generation and provided evidence of spatial localization of clot formation by TM with final clot lengths of 0.2-2 mm under diffusive conditions.

These studies provide an interesting analysis of the roles of specific factors in relation to space due to diffusive effects, but neglect the essential role of blood flow in the transport analysis. Additionally, the spatial dynamics of clot localization by thrombomodulin would likely be affected by restricting the inhibitor to its physiologic site on the vessel surface.

Finite Element Modeling

Finite element method (FEM) is a numerical technique for solving partial differential equations. Originally proposed in the 1940s to approach structural analysis problems in civil engineering, FEM now finds application in a wide variety of disciplines. The computational method relies on mesh discretization of a continuous domain which subdivides the space into a finite number of ‘elements’. The physics of each element are defined by its own set of physical properties and boundary conditions, and the simultaneous solution of the equations describing the individual elements approximate the behavior of the overall domain.

Sumanas W. Jordan, PhD Thesis. A Mathematical Model of Tissue Factor-Induced Blood Coagulation: Discrete Sites of Initiation and Regulation under Conditions of Flow.

Doctor of Philosophy in Biomedical Engineering. Emory University, Georgia Institute of Technology. May 2010.  Under supervision of: Dr. Elliot L. Chaikof, Departments of Surgery and Biomedical Engineering.

Blood Coagulation (Thrombin) and Protein C Pat...

Blood Coagulation (Thrombin) and Protein C Pathways (Blood_Coagulation_and_Protein_C_Pathways.jpg) (Photo credit: Wikipedia)

Coagulation cascade

Coagulation cascade (Photo credit: Wikipedia)

 

Cardiovascular Physiology: Modeling, Estimation and Signal Processing

With cardiovascular diseases being among the main causes of death in the world, quantitative modeling, assessment and monitoring of cardiovascular dynamics, and functioning play a critical role in bringing important breakthroughs to cardiovascular care. Quantification of cardiovascular physiology and its control mechanisms from physiological recordings, by use of mathematical models and algorithms, has been proved to be of important value in understanding the causes of cardiovascular diseases and assisting the diagnostic and prognostic process. This E-Book is derived from the Frontiers in Computational Physiology and Medicine Research Topic entitled “Engineering Approaches to Study Cardiovascular Physiology: Modeling, Estimation and Signal Processing.”

There are two review articles. The first review article by Chen et al. (2012) presents a unified point process probabilistic framework to assess heart beat dynamics and autonomic cardiovascular control. Using clinical recordings of healthy subjects during Propofol anesthesia, the authors demonstrate the effectiveness of their approach by applying the proposed paradigm to estimate

  • instantaneous heart rate (HR),
  • heart rate variability (HRV),
  • respiratory sinus arrhythmia (RSA)
  • baroreflex sensitivity (BRS).

The second review article, contributed by Zhang et al. (2011), provides a comprehensive overview of tube-load model parameter estimation for monitoring arterial hemodynamics.

The remaining eight original research articles can be mainly classified into two categories. The two articles from the first category emphasize modeling and estimation methods. In particular, the paper “Modeling the autonomic and metabolic effects of obstructive sleep apnea: a simulation study” by Cheng and Khoo (2012), combines computational modeling and simulations to study the autonomic and metabolic effects of obstructive sleep apnea (OSA).

The second paper, “Estimation of cardiac output and peripheral resistance using square-wave-approximated aortic flow signal” by Fazeli and Hahn (2012), presents a model-based approach to estimate cardiac output (CO) and total peripheral resistance (TPR), and validates the proposed approach via in vivo experimental data from animal subjects.

The six articles in the second category focus on application of signal processing techniques and statistical tools to analyze cardiovascular or physiological signals in practical applications. the paper “Modulation of the sympatho-vagal balance during sleep: frequency domain study of heart rate variability and respiration” by Cabiddu et al. (2012), uses spectral and cross-spectral analysis of heartbeat and respiration signals to assess autonomic cardiac regulation and cardiopulmonary coupling variations during different sleep stages in healthy subjects.

The paper “increased non-gaussianity of heart rate variability predicts cardiac mortality after an acute myocardial infarction” by Hayano et al. (2011) uses a new non-gaussian index to assess the HRV of cardiac mortality using 670 post-acute myocardial infarction (AMI) patients. the paper “non-gaussianity of low frequency heart rate variability and sympathetic activation: lack of increases in multiple system atrophy and parkinson disease” by Kiyono et al. (2012), applies a non-gaussian index to assess HRV in patients with multiple system atrophy (MSA) and parkinson diseases and reports the relation between the non-gaussian intermittency of the heartbeat and increased sympathetic activity. The paper “Information domain approach to the investigation of cardio-vascular, cardio-pulmonary, and vasculo-pulmonary causal couplings” by Faes et al. (2011), proposes an information domain approach to evaluate nonlinear causality among heartbeat, arterial pressure, and respiration measures during tilt testing and paced breathing protocols. The paper “integrated central-autonomic multifractal complexity in the heart rate variability of healthy humans” by Lin and Sharif (2012), uses a relative multifractal complexity measure to assess HRV in healthy humans and discusses the related implications in central autonomic interactions. Lastly, the paper “Time scales of autonomic information flow in near-term fetal sheep” by Frasch et al. (2012), analyzes the autonomic information flow (AIF) with kullback–leibler entropy in fetal sheep as a function of vagal and sympathetic modulation of fetal HRV during atropine and propranolol blockade.

In summary, this Research Topic attempts to give a general panorama of the possible state-of-the-art modeling methodologies, practical tools in signal processing and estimation, as well as several important clinical applications, which can altogether help deepen our understanding about heart physiology and pathology and further lead to new scientific findings. We hope that the readership of Frontiers will appreciate this collected volume and enjoy reading the presented contributions. Finally, we are grateful to all contributed authors, reviewers, and editorial staffs who had all put tremendous effort to make this E-Book a reality.

Cabiddu, R., Cerutti, S., Viardot, G., Werner, S., and Bianchi, A. M. (2012). Modulation of the sympatho-vagal balance during sleep: frequency domain study of heart rate variability and respiration. Front. Physio. 3:45. doi: 10.3389/fphys.2012.00045

Chen, Z., Purdon, P. L., Brown, E. N., and Barbieri, R. (2012). A unified point process probabilistic framework to assess heartbeat dynamics and autonomic cardiovascular control. Front. Physio. 3:4. doi: 10.3389/fphys.2012.00004

Cheng, L., and Khoo, M. C. K. (2012). Modeling the autonomic and metabolic effects of obstructive sleep apnea: a simulation study. Front. Physio. 2:111. doi: 10.3389/fphys.2011.00111

Faes, L., Nollo, G., and Porta, A. (2011). Information domain approach to the investigation of cardio-vascular, cardio-pulmonary, and vasculo-pulmonary causal couplings. Front. Physio. 2:80. doi: 10.3389/fphys.2011.00080

Fazeli, N., and Hahn, J.-O. (2012). Estimation of cardiac output and peripheral resistance using square-wave-approximated aortic flow signal. Front. Physio. 3:298. doi: 10.3389/fphys.2012.00298

Frasch, M. G., Frank, B., Last, M., and Müller, T. (2012). Time scales of autonomic information flow in near-term fetal sheep. Front. Physio. 3:378. doi: 10.3389/fphys.2012.00378

Hayano, J., Kiyono, K., Struzik, Z. R., Yamamoto, Y., Watanabe, E., Stein, P. K., et al. (2011). Increased non-gaussianity of heart rate variability predicts cardiac mortality after an acute myocardial infarction. Front. Physio. 2:65. doi: 10.3389/fphys.2011.00065

Kiyono, K., Hayano, J., Kwak, S., Watanabe, E., and Yamamoto, Y. (2012). Non-Gaussianity of low frequency heart rate variability and sympathetic activation: lack of increases in multiple system atrophy and Parkinson disease. Front. Physio. 3:34. doi: 10.3389/fphys.2012.00034

Lin, D. C., and Sharif, A. (2012). Integrated central-autonomic multifractal complexity in the heart rate variability of healthy humans. Front. Physio. 2:123. doi: 10.3389/fphys.2011.00123

Zhang, G., Hahn, J., and Mukkamala, R. (2011). Tube-load model parameter estimation for monitoring arterial hemodynamics. Front. Physio. 2:72. doi: 10.3389/fphys.2011.00072

Citation: Chen Z and Barbieri R (2012) Editorial: engineering approaches to study cardiovascular physiology: modeling, estimation, and signal processing. Front. Physio. 3:425. doi: 10.3389/fphys.2012.00425

fluctuations of cerebral blood flow and metabolic demand following hypoxia in neonatal brain

Most of the research investigating the pathogenesis of perinatal brain injury following hypoxia-ischemia has focused on excitotoxicity, oxidative stress and an inflammatory response, with the response of the developing cerebrovasculature receiving less attention. This is surprising as the presentation of devastating and permanent injury such as germinal matrix-intraventricular haemorrhage (GM-IVH) and perinatal stroke are of vascular origin, and the origin of periventricular leukomalacia (PVL) may also arise from poor perfusion of the white matter. This highlights that cerebrovasculature injury following hypoxia could primarily be responsible for the injury seen in the brain of many infants diagnosed with hypoxic-ischemic encephalopathy (HIE).

The highly dynamic nature of the cerebral blood vessels in the fetus, and the fluctuations of cerebral blood flow and metabolic demand that occur following hypoxia suggest that the response of blood vessels could explain both regional protection and vulnerability in the developing brain.

This review discusses the current concepts on the pathogenesis of perinatal brain injury, the development of the fetal cerebrovasculature and the blood brain barrier (BBB), and key mediators involved with the response of cerebral blood vessels to hypoxia.

Baburamani AA, Ek CJ, Walker DW and Castillo-Melendez M. Vulnerability of the developing brain to hypoxic-ischemic damage: contribution of the cerebral vasculature to injury and repair? Front. Physio. 2012;  3:424. doi: 10.3389/fphys.2012.00424

remodeling of coronary and cerebral arteries and arterioles 

Effects of hypertension on arteries and arterioles often manifest first as a thickened wall, with associated changes in passive material properties (e.g., stiffness) or function (e.g., cellular phenotype, synthesis and removal rates, and vasomotor responsiveness). Less is known, however, regarding the relative evolution of such changes in vessels from different vascular beds.

We used an aortic coarctation model of hypertension in the mini-pig to elucidate spatiotemporal changes in geometry and wall composition (including layer-specific thicknesses as well as presence of collagen, elastin, smooth muscle, endothelial, macrophage, and hematopoietic cells) in three different arterial beds, specifically aortic, cerebral, and coronary, and vasodilator function in two different arteriolar beds, the cerebral and coronary.

Marked geometric and structural changes occurred in the thoracic aorta and left anterior descending coronary artery within 2 weeks of the establishment of hypertension and continued to increase over the 8-week study period. In contrast, no significant changes were observed in the middle cerebral arteries from the same animals. Consistent with these differential findings at the arterial level, we also found a diminished nitric oxide-mediated dilation to adenosine at 8 weeks of hypertension in coronary arterioles, but not cerebral arterioles.

These findings, coupled with the observation that temporal changes in wall constituents and the presence of macrophages differed significantly between the thoracic aorta and coronary arteries, confirm a strong differential progressive remodeling within different vascular beds.

These results suggest a spatiotemporal progression of vascular remodeling, beginning first in large elastic arteries and delayed in distal vessels.

Hayenga HN, Hu J-J, Meyer CA, Wilson E, Hein TW, Kuo L and Humphrey JD  Differential progressive remodeling of coronary and cerebral arteries and arterioles in an aortic coarctation model of hypertension. Front. Physio. 2012; 3:420. doi: 10.3389/fphys.2012.00420

C-reactive protein oxidant-mediated release of pro-thrombotic  factor

Inflammation and the generation of reactive oxygen species (ROS) have been implicated in the initiation and progression of atherosclerosis. Although C-reactive protein (CRP) has traditionally been considered to be a biomarker of inflammation, recent in vitro and in vivo studies have provided evidence that CRP, itself, exerts pro-thrombotic effects on vascular cells and may thus play a critical role in the development of atherothrombosis. Of particular importance is that CRP interacts with Fcγ receptors on cells of the vascular wall giving rise to the release of pro-thrombotic factors. The present review focuses on distinct sources of CRP-mediated ROS generation as well as the pivotal role of ROS in CRP-induced tissue factor expression. These studies provide considerable insight into the role of the oxidative mechanisms in CRP-mediated stimulation of pro-thrombotic factors and activation of platelets. Collectively, the available data provide strong support for ROS playing an important intermediary role in the relationship between CRP and atherothrombosis.

Zhang Z, Yang Y, Hill MA and Wu J.  Does C-reactive protein contribute to atherothrombosis via oxidant-mediated release of pro-thrombotic factors and activation of platelets? Front. Physio.  2012; 3:433. doi: 10.3389/fphys.2012.00433

CRP association with Peripheral Vascular Disease

To determine whether the increase in plasma levels of C-Reactive Protein (CRP), a non-specifi c reactant in the acute-phase of systemic infl ammation, is associated with clinical severity of peripheral arterial disease (PAD).

This is a cross-sectional study at a referral hospital center of institutional practice in Madrid, Spain.  These investigators took a stratifi ed random sampling of 3370 patients with symptomatic PAD from the outpatient vascular laboratory database in 2007 in the order of their clinical severity:

  • the fi rst group of patients with mild chronological clinical severity who did not require surgical revascularization,
  • the second group consisted of patients with moderate clinical severity who had only undergone only one surgical revascularization procedure and
  • the third group consisted of patients who were severely affected and had undergone two or more surgical revascularization procedures of the lower extremities in different areas or needed late re-interventions.

The Neyman affi xation was used to calculate the sample size with a fi xed relative error of 0.1.

A homogeneity analysis between groups and a unifactorial analysis of comparison of medians for CRP was done.

The groups were homogeneous for

  • age
  • smoking status
  • Arterial Hypertension
  • diabetes mellitus
  • dyslipemia
  • homocysteinemia and
  • specifi c markers of infl ammation.

In the unifactorial analysis of multiple comparisons of medians according to Scheffé, it was observed that

the median values of CRP plasma levels were increased in association with higher clinical severity of PAD

  • 3.81 mg/L [2.14–5.48] vs.
  • 8.33 [4.38–9.19] vs.
  • 12.83 [9.5–14.16]; p  0.05

as a unique factor of tested ones.

Plasma levels of CRP are associated with not only the presence of atherosclerosis but also with its chronological clinical severity.

De Haro J, Acin F, Medina FJ, Lopez-Quintana A, and  March JR.  Relationship Between the Plasma Concentration of C-Reactive Protein and Severity of Peripheral Arterial Disease.
Clinical Medicine: Cardiology 2009;3: 1–7

Hemostasis induced by hyperhomocysteinemia

Elevated concentration of homocysteine (Hcy) in human tissues, defined as hyperhomocysteinemia has been correlated with some diseases, such as

  • cardiovascular
  • neurodegenerative
  • kidney disorders

L-Homocysteine (Hcy) is an endogenous amino acid, containing a free thiol group, which in healthy cells is involved in methionine and cysteine synthesis/resynthesis. Indirectly, Hcy participates in methyl, folate, and cellular thiol metabolism. Approximately 80% of total plasma Hcy is protein-bound, and only a small amount exists as a free reduced Hcy (about 0.1 μM). The majority of the unbound fraction of Hcy is oxidized, and forms dimers (homocystine) or mixed disulphides consisting of cysteine and Hcy.

Two main pathways of Hcy biotoxicity are discussed:

  1. Hcy-dependent oxidative stress – generated during oxidation of the free thiol group of Hcy. Hcy binds via a disulphide bridge with

—     plasma proteins

—     or with other low-molecular plasma  thiols

—     or with a second Hcy molecule.

Accumulation of oxidized biomolecules alters the biological functions of many cellular pathways.

  1. Hcy-induced protein structure modifications, named homocysteinylation.

Two main types of homocysteinylation exist: S-homocysteinylation and N-homocysteinylation; both considered as posttranslational protein modifications.

a)      S-homocysteinylation occurs when Hcy reacts, by its free thiol group, with another free thiol derived from a cysteine residue in a protein molecule.

These changes can alter the thiol-dependent redox status of proteins.

b)      N-homocysteinylation takes place after acylation of the free ε-amino lysine groups of proteins by the most reactive form of Hcy — its cyclic thioester (Hcy thiolactone — HTL), representing up to 0.29% of total plasma Hcy.

Homocysteine occurs in human blood plasma in several forms, including the most reactive one, the homocysteine thiolactone (HTL) — a cyclic thioester, which represents up to 0.29% of total plasma Hcy. In human blood, N-homocysteinylated (N-Hcy-protein) and S-homocysteinylated proteins (S-Hcy-protein) such as NHcy-hemoglobin, N-(Hcy-S-S-Cys)-albumin, and S-Hcyalbumin are known. Other pathways of Hcy biotoxicity might be apoptosis and excitotoxicity mediated through glutamate receptors. The relationship between homocysteine and risk appears to hold for total plasma concentrations of homocysteine between 10 and 30 μM.

Different forms of homocysteine present in human blood.

*Total level of homocysteine — the term “total homocysteine” describes the pool of homocysteine released by reduction of all disulphide bonds in the sample (Perla-Kajan et al., 2007; Zimny, 2008; Manolescu et al., 2010, modified).

The form of Hcy The concentration in human blood
Homocysteine thiolactone (HTL) 0–35 nM
Protein N-linked homocysteine:
N-Hcy-hemoglobin, N-(Hcy-S-S-Cys)-albumin
about 15.5 μM: 12.7 μM, 2.8 μM
Protein S-linked homocysteine — S-Hcy-albumin about 7.3 μM*
Homocystine (Hcy-S-S-Hcy) and combined with cysteine to from mixed disulphides (Hcy-S-S-Cys) about 2 μM*
Free reduced Hcy about 0.1 μM*

As early as in the 1960s it was noted that the risk of atherosclerosis is markedly increased in patients with homocystinuria, an inherited disease resulting from homozygous CBS deficiency and characterized by episodes of

—     thromboembolism

—     mental retardation

—     lens dislocation

—     hepatic steatosis

—     osteoporosis.

—     very high concentrations of plasma homocysteine and methionine.

Patients with homocystinuria have very severe hyperhomocysteinemia, with plasma homocysteine concentration reaching even 400 μM, and represent a very small proportion of the population (approximately 1 in 200,000 individuals). Heterozygous lack of CBS, CBS mutations and polymorphism of the methylenetetrahydrofolate reductase gene are considered to be the most probable causes of hyperhomocysteinemia.

The effects of hyperhomocysteinemia include the complex process of hemostasis, which regulates the properties of blood flow. Interactions of homocysteine and its different derivatives, including homocysteine thiolactone, with the major components of hemostasis are:

  • endothelial cells
  • platelets
  • fibrinogen
  • plasminogen

Elevated plasma Hcy (>15 μM; Hcy) is associated with an increased risk of cardiovascular diseases

  • thrombosis
  • thrombosis related diseases
  • ischemic brain stroke (independent of other, conventional risk factors of this disease)

Every increase of 2.5 μM in plasma Hcy may be associated with an increase of stroke risk of about 20%.  Total plasma Hcy level above 20 μM are associated with a nine-fold increase of the myocardial infarction and stroke risk, in comparison to the concentrations below 9 μM. The increase of Hcy concentration has been also found in other human pathologies, including neurodegenerative diseases

Modifications of hemostatic proteins (N-homocysteinylation or S-homocysteinylation) induced by Hcy or its thiolactone seem to be the main cause of homocysteine biotoxicity in hemostatic abnormalities.

Hcy and HTL may act as oxidants, but various polyphenolic antioxidants are able to inhibit the oxidative damage induced by Hcy or HTL. Therefore, we have to consider the role of phenolic antioxidants in hyperhomocysteinemia –induced changes in hemostasis.

The synthesis of homocysteine thiolactone is associated with the activation of the amino acid by aminoacyl-tRNA synthetase (AARS). Hcy may also undergo erroneous activation, e.g. by methionyl-t-RNA synthetase (MetRS). In the first step of conversion of Hcy to HTL, MetRS misactivates Hcy giving rise to homocysteinyl-adenylate. In the next phase, the homocysteine side chain thiol group reacts with the activated carboxyl group and HTL is produced. The level of HTL synthesis in cultured cells depends on Hcy and Met levels.

Hyperhomocysteinemia and Changes in Fibrinolysis and Coagulation Process

The fibrinolytic activity of blood is regulated by specific inhibitors; the inhibition of fibrinolysis takes place at the level of plasminogen activation (by PA-inhibitors: plasminogen activator inhibitor type-1, -2; PAI-1 or PAI-2) or at the level of plasmin activity (mainly by α2-antiplasmin). Hyperhomocysteinemia disturbs hemostasis and shifts the hemostatic mechanisms in favor of thrombosis. The recent reports indicate that the prothrombotic state observed in hyperhomocysteinemia may arise not only due to endothelium dysfunction or blood platelet and coagulation activation, but also due to impaired fibrinolysis. Hcy-modified fibrinogen is more resistant to the fibrinolytic action. Oral methionine load increases total Hcy, but may diminish the fibrinolytic activity of the euglobulin plasma fraction. Homocysteine-lowering therapies may increase fibrinolytic activity, thereby, prevent atherothrombotic events in patients with cardiovascular diseases after the first myocardial infarction.

Homocysteine — Fibronectin Interaction and its Consequences

Fibronectin (Fn) plays key roles in

  • cell adhesion
  • migration
  • embryogenesis
  • differentiation
  • hemostasis
  • thrombosis
  • wound healing
  • tissue remodeling

Interaction of FN with fibrin, mediated by factor XIII transglutaminase, is thought to be important for cell adhesion or cell migration into fibrin clots. After tissue injury, a blood clot formation serves the dual role of restoring vascular integrity and serving as a temporary scaffold for the wound healing process. Fibrin and plasma FN, the major protein components of blood clots, are essential to perform these functions. In the blood clotting process, after fibrin deposition, plasma FN-fibrin matrix is covalently crosslinked, and it then promotes fibroblast adhesion, spreading, and migration into the clot.

Homocysteine binds to several human plasma proteins, including fibronectin. If homocysteine binds to fibronectin via a disulphide linkage, this binding results in a functional change, namely, the inhibition of fibrin binding by fibronectin. This inhibition may lead to a prolonged recovery from a thrombotic event and contribute to vascular occlusion.

Grape seeds are one of the richest plant sources of phenolic substances, and grape seed extract reduces the toxic effect of Hcys and HTL on fibrinolysis. The grape seed extract (12.5–50 μg/ml) supported plasminogen to plasmin conversion inhibited by Hcys or HTL. In vitro experiments showed in the presence of grape seed extract (at the highest tested concentration — 50 μg/ml) the increase of about 78% (for human plasminogen-treated with Hcys) and 56% (for human plasma-treated with Hcys). Thus, in the in vitro model system, that the grape seed extract (12.5–50 μg/ml) diminished the reduction of thiol groups and of lysine ε-amino groups in plasma proteins treated with Hcys (0.1 mM) or HTL (1 μM). In the presence of the grape seed extract at the concentration of 50 μg/ml, the level of reduction of thiol groups reached about 45% (for plasma treated with Hcys) and about 15% (for plasma treated with HTL).

In the presence of the grape seed extract at the concentration of 50 μg/ml, the level of reduction of thiol groups reached about 45% (for plasma treated with Hcys) and about 15% (for plasma treated with HTL).Very similar protective effects of the grape seed extract were observed in the measurements of lysine ε-amino groups in plasma proteins treated with Hcys or HTL. These results indicated that the extract from berries of Aronia melanocarpa (a rich source of phenolic substances) reduces the toxic effects of Hcy and HTL on the hemostatic properties of fibrinogen and plasma. These findings indicate a possible protective action of the A. melanocarpa extract in hyperhomocysteinemia-induced cardiovascular disorders. Moreover, the extract from berries of A. melanocarpa, due to its antioxidant action, significantly attenuated the oxidative stress (assessed by measuring of the total antioxidant status — TAS) in plasma in a model of hyperhomocysteinemia.

Proposed model for the protective role of phenolic antioxidants on selected elements of hemostasis during hyperhomocysteinemia.

various antioxidants (present in human diet), including phenolic compounds, may reduce the toxic effects of Hcy or its derivatives on hemostasis. These findings give hope for the develop development of dietary supplements, which will be capable of preventing thrombosis which occurs under pathological conditions, observed also in hyperhomocysteinemia, such as plasma procoagulant activity and oxidative stress.

Malinowska J,  Kolodziejczyk J and Olas B. The disturbance of hemostasis induced by hyper-homocysteinemia; the role of antioxidants. Acta Biochimica Polonica 2012; 59(2): 185–194.

Lipoprotein (a)

Lipoprotein (a) (Lp(a)), for the first time described in 1963 by Berg belongs to the lipoproteins with the strongest atherogenic effect. Its importance for the development of various atherosclerotic vasculopathies (coronary heart disease, ischemic stroke, peripheral vasculopathy, abdominal aneurysm) was recognized considerably later.

Lipoprotein(a) (Lp(a)), an established risk marker of cardiovascular diseases, is independent from other risk markers. The main difference of Lp(a) compared to low density lipoprotein (LDL) is the apo(a) residue, covalently bound to apoB is covalently by a disulfide-bridge. Apo(a) synthesis is performed in the liver, probably followed by extracellular assembly to the apoB location of the LDL.

 

ApoB-100_______LDL¬¬___ S-S –    9

Apo(a) has been detected bound to triglyceride-rich lipoproteins (Very Low Density Lipoproteins; VLDL). Corresponding to the structural similarity to LDL, both particles are very similar to each other with regard to their composition. It is a glycoprotein which underlies a large genetic polymorphism caused by a variation of the kringle-IV-type-2 repeats of the protein, characterized by a structural homology to plasminogen. Apo(a)’s structural homology to plasminogen, shares the gene localization on chromosome 6. The kringle repeats present a particularly characteristic structure, which have a high similarity to kringle IV (K IV) of plasminogen. Apo(a) also has a kringle V structure of plasminogen and also a protease domain, which cannot be activated, as opposed to the one of plasminogen. At least 30 genetically determined apo(a) isoforms were identified in man.

Features:

  • Non covalent binding of kringle -4 types 7 and 8 of apo (a) to apo B
  • Disulfide bond at Cys4326 of ApoB (near its receptor binding domain ) and the only free cysteine group in K –IV type 9 (Cys4057) of apo(a )
  • Binding to fibrin and cell membranes
  • Enhancement by small isoforms ; high concentrations compared to plasminogen and homocysteine
  • Binding to different lysine rich components of the coagulation system (e. g. TFPI)
  • Intense homology to plasminogen but no protease activity
ApoB-100_______LDL¬¬___ S-S – 9

The synthesis of Lp(a), which thus occurs as part of an assembly, is a two-step process.

  • In a first step, which can be competitively inhibited by lysine analogues, the free sulfhydryl groups of apo(a) and apoB are brought close together.
  • The binding of apo(a) then occurs near the apoB domain which binds to the LDL receptor, resulting in a reduced affinity of Lp(a) to the LDL-receptor.

Particles that show a reduced affinity to the LDL receptor are not able to form stable compounds with apo(a). Thus the largest part of apo(a) is present as apo(a) bound to LDL. Only a small, quantitatively variable part of apo(a) remains as free apo(a) and probably plays an important role in the metabolism and physiological function of Lp(a).

The Lp(a) plasma concentration in the population is highly skewed and determined to more than 90 % by genetic factors. In healthy subjects the Lp(a)-concentration is correlated with its synthesis.

It is assumed that the kidney has a specific function in Lp(a) catabolizm, since nephrotic syndrome and terminal kidney failure are associated with an elevation of the Lp(a) plasma concentration. One consequence of the poor knowledge of the metabolic path of Lp(a) is the fact that so far pharmaceutical science has failed to develop drugs that are able to reduce elevated Lp(a) plasma concentrations to a desirable level.

Plasma concentrations of Lp(a) are affected by different diseases (e.g. diseases of liver and kidney), hormonal factors (e.g. sexual steroids, glucocorticoids, thyroid hormones), individual and environmental factors (e.g. age, cigarette smoking) as well as pharmaceuticals (e.g. derivatives of nicotinic acid) and therapeutic procedures (lipid apheresis). This review describes the physiological regulation of Lp(a) as well as factors influencing its plasma concentration.

Apart from its significance as an important agent in the development of atherosclerosis, Lp(a) has even more physiological functions, e.g. in

  • wound healing
  • angiogenesis
  • hemostasis

However, in the meaning of a pleiotropic mechanism the favorable action mechanisms are opposed by pathogenic mechanisms, whereby the importance of Lp(a) in atherogenesis is stressed.

Lp(a) in Atherosclerosis

In transgenic, hyperlipidemic and Lp(a) expressing Watanabe rabbits, Lp(a) leads to enhanced atherosclerosis. Under the influence of Lp(a), the binding of Lp(a) to glycoproteins, e.g. laminin, results – via its apo(a)-part – both in

  • an increased invasion of inflammatory cells and in
  • an activation of smooth vascular muscle cells

with subsequent calcifications in the vascular wall.

The inhibition of transforming growth factor-β1 (TGF-β1) activation is another mechanism via which Lp(a) contributes to the development of atherosclerotic vasculopathies. TGF-β1 is subject to proteolytic activation by plasmin and its active form leads to an inhibition of the proliferation and migration of smooth muscle cells, which play a central role in the formation and progression of atherosclerotic vascular diseases.

In man, Lp(a) is an important risk marker which is independent of other risk markers. Its importance, partly also under consideration of the molecular weight and other genetic polymorphisms, could be demonstrated by a high number of epidemiological and clinical studies investigating the formation and progression of atherosclerosis, myocardial infarction, and stroke.

Lp(a) in Hemostasis

Lp(a) is able to competitively inhibit the binding of plasminogen to fibrinogen and fibrin, and to inhibit the fibrin-dependent activation of plasminogen to plasmin via the tissue plasminogen activator, whereby apo(a) isoforms of low molecular weight have a higher affinity to fibrin than apo(a) isoforms of higher molecular weight. Like other compounds containing sulfhydryl groups, homocysteine enhances the binding of Lp(a) to fibrin.

Pleiotropic effect of Lp(a).

Prothrombotic :

  • Binding to fibrin
  • Competitive inhibition of plasminogen
  • Stimulation of plasminogen activator inhibitor I and II (PAI -I, PAI -II)
  • Inactivation of tissue factor pathway inhibitor (TFPI)

Antithrombotic :

  • Inhibition of platelet activating factor acetylhydrolase (PAF -AH)
  • Inhibition of platelet activating factor
  • Inhibition of collagen dependent platelet aggregation
  • Inhibition of secretion of serotonin und thromboxane

Lp(a) in Angiogenesis

Lp(a) is also important for the process of angiogenesis and the sprouting of new vessels.

  • angiogenesis starts with the remodelling of matrix proteins and
  • activation of matrix metalloproteinases (MMP).

The latter ones are usually synthesised as

  • inactive zymogens and
  • require activation by proteases,

Recall that Apo(a) is not activated by proteases. The angiogenesis is also accomplished by plasminogen. Lp(a) and apo(a) and its fragments has an antiangiogenetic and metastasis inhibiting effect related to the structural homology with plasminogen without the protease activity.

Siekmeier R, Scharnagl H, Kostner GM, T. Grammer T, Stojakovic T and März W.  Variation of Lp(a) Plasma Concentrations in Health and Disease.  The Open Clinical Chemistry Journal, 2010; 3: 72-89.

LDL-Apheresis

In 1985, Brown and Goldstein were awarded the Nobel Prize for medicine for their work on the regulation of cholesterol metabolism. On the basis of numerous studies, they were able to demonstrate that circulating low-density lipoprotein (LDL) is absorbed into the cell through receptor linked endocytosis. The absorption of LDL into the cell is specific and is mediated by a LDL receptor. In patients with familial hypercholesterolemia, this receptor is changed, and the LDL particles can no longer be recognized. Their absorption can thus no longer be mediated, leading to an accumulation of LDL in blood.

Furthermore, an excess supply of cholesterol also blocks the 3-hydrox-3 methylglutaryl-Co enzyme A (HMG CoA), reductase enzyme, which otherwise inhibits the cholesterol synthesis rate. Brown and Goldstein also determined the structure of the LDL receptor. They discovered structural defects in this receptor in many patients with familial hypercholesterolemia. Thus, familial hypercholesterolemia was the first metabolic disease that could be tracked back to the mutation of a receptor gene.

Dyslipoproteinemia in combination with diabetes mellitus causes a cumulative insult to the vasculature resulting in more severe disease which occurs at an earlier age in large and small vessels as well as capillaries. The most common clinical conditions resulting from this combination are myocardial infarction and lower extremity vascular disease. Ceriello et al. show an independent and cumulative effect of postprandial hypertriglyceridemia and hyperglycemia on endothelial function, suggesting oxidative stress as common mediator of such effect. The combination produces greater morbidity and mortality than either alone.

As an antiatherogenic factor, HDL cholesterol correlates inversely to the extent of postprandial lipemia. A high concentration of HDL is a sign that triglyceride-rich particles are quickly decomposed in the postprandial phase of lipemia. Conversely, with a low HDL concentration this decomposition is delayed. Thus, excessively high triglyceride concentrations are accompanied by very low HDL counts. This combination has also been associated with an increased risk of pancreatitis.

The importance of lipoprotein (a) (Lp(a)) as an atherogenic substance has also been recognized in recent years. Lp(a) is very similar to LDL. But it also contains Apo(a), which is very similar to plasminogen, enabling Lp(a) to bind to fibrin clots. Binding of plasminogen is prevented and fibrinolysis obstructed. Thrombi are integrated into the walls of the arteries and become plaque components.

Another strong risk factor for accelerated atherogenesis, which must be mentioned here, are the widespread high homocysteine levels found in dialysis patients. This risk factor is independent of classic risk factors such as high cholesterol and LDL levels, smoking, hypertension, and obesity, and much more predictive of coronary events in dialysis patients than are these better-known factors. Homocysteine is a sulfur aminoacid produced in the metabolism of methionine. Under normal conditions, about 50 percent of homocysteine is remethylated to methionine and the remaining via the transsulfuration pathway.

Defining hyperhomocysteinemia as levels greater than the 90th percentile of controls and elevated Lp(a) level as greater than 30mg/dL, the frequency of the combination increased with declining renal function. Fifty-eight percent of patients with a GFR less than 10mL/min had both hyperhomocysteinemia and elevated Lp(a) levels, and even in patients with mild renal impairment, 20 percent of patients had both risk factors present.

The prognosis of patients suffering from severe hyperlipidemia, sometimes combined with elevated lipoprotein (a) levels, and coronary heart disease refractory to diet and lipid-lowering drugs is poor. For such patients, regular treatment with low-density lipoprotein (LDL) apheresis is the therapeutic option. Today, there are five different LDL-apheresis systems available: cascade filtration or lipid filtration, immunoadsorption, heparin-induced LDL precipitation, dextran sulfate LDL adsorption, and the LDL hemoperfusion. The requirement that the original level of cholesterol is to be reduced by at least 60 percent is fulfilled by all these systems.

There is a strong correlation between hyperlipidemia and atherosclerosis. Besides the elimination of other risk factors, in severe hyperlipidemia therapeutic strategies should focus on a drastic reduction of serum lipoproteins. Despite maximum conventional therapy with a combination of different kinds of lipid-lowering drugs, sometimes the goal of therapy cannot be reached. Hence, in such patients, treatment with LDL-apheresis is indicated. Technical and clinical aspects of these five different LDL-apheresis methods are depicted. There were no significant differences with respect to or concerning all cholesterols, or triglycerides observed.

High plasma levels of Lp(a) are associated with an increased risk for atherosclerotic coronary heart       disease
(CHD) by a mechanism yet to be determined. Because of its structural properties, Lp(a) can have both atherogenic and thrombogenic potentials. The means for correcting the high plasma levels of Lp(a) are still limited in effectiveness. All drug therapies tried thus far have failed. The most effective therapeutic methods in lowering Lp(a) are the LDL-apheresismethods. Since 1993, special immunoadsorption polyclonal antibody columns (Pocard, Moscow, Russia) containing sepharose bound anti-Lp(a) have been available for the treatment of patients with elevated Lp(a) serum concentrations.

With respect to elevated lipoprotein (a) levels, however, the immunoadsorption method seems to be most effective. The different published data clearly demonstrate that treatment with LDL-apheresis in patients suffering from severe hyperlipidemia refractory to maximum conservative therapy is effective and safe in long-term application.

LDL-apheresis decreases not only LDL mass but also improves the patient’s life expectancy. LDL-apheresis performed with different techniques decreases the susceptibility of LDL to oxidation. This decrease may be related to a temporary mass imbalance between freshly produced and older LDL particles. Furthermore, the baseline fatty acid pattern influences pretreatment and postreatment susceptibility to oxidation.

Bambauer R, Bambauer C, Lehmann B, Latza R, and Ralf Schiel R. LDL-Apheresis: Technical and Clinical Aspects. The Scientific World Journal 2012; Article ID 314283, pp 1-19. doi:10.1100/2012/314283

Summary:  This discussion is a two part sequence that first establishes the known strong relationship between blood flow viscosity, shear stress, and plasma triglycerides (VLDL) as risk factors for hemostatic disorders leading to thromboembolic disease, and the association with atherosclerotic disease affecting the heart, the brain (via carotid blood flow), peripheral circulation,the kidneys, and retinopathy as well.

The second part discusses the modeling of hemostasis and takes into account the effects of plasma proteins involved with red cell and endothelial interaction, which is related to part I.  The current laboratory assessment of thrombophilias is taken from a consensus document of the American Society for Clinical Pathology.  The problems encountered are sufficient for the most common problems of coagulation testing and monitoring, but don’t address the large number of patients who are at risk for complications of accelerated vasoconstrictive systemic disease that precede serious hemostatic problems.  Special attention is given to Lp(a) and to homocysteine.  Lp(a) is a protein that has both prothrombotic and antithrombotic characteristics, and is a homologue of plasminogen and is composed of an apo(a) bound to LDL.  Unlike plasminogen, it has no protease activity.   Homocysteine elevation is a known risk factor for downstream myocardial infarct.  Homocysteine is a mirror into sulfur metabolism, so an increase is an independent predictor of risk, not fully discussed here.  The modification of risk is discussed by diet modification.  In the most serious cases of lipoprotein disorders, often including Lp(a) the long term use of LDL-apheresis is described.

see Relevent article that appears in NEJM from American College of Cardiology

Apolipoprotein(a) Genetic Sequence Variants Associated With Systemic Atherosclerosis and Coronary Atherosclerotic Burden but Not With Venous Thromboembolism

Helgadottir A, Gretarsdottir S, Thorleifsson G, et al

J Am Coll Cardiol. 2012;60:722-729

Study Summary

The LPA gene codes for apolipoprotein(a), which, when linked with low-density lipoprotein particles, forms lipoprotein(a) [Lp(a)] — a well-studied molecule associated with coronary artery disease (CAD). The Lp(a) molecule has both atherogenic and thrombogenic effects in vitro , but the extent to which these translate to differences in how atherothrombotic disease presents is unknown.

LPA contains many single-nucleotide polymorphisms, and 2 have been identified by previous groups as being strongly associated with levels of Lp(a) and, as a consequence, strongly associated with CAD. However, because atherosclerosis is thought to be a systemic disease, it is unclear to what extent Lp(a) leads to atherosclerosis in other arterial beds (eg, carotid, abdominal aorta, and lower extremity), as well as to other thrombotic disorders (eg, ischemic/cardioembolic stroke and venous thromboembolism). Such distinctions are important, because therapies that might lower Lp(a) could potentially reduce forms of atherosclerosis beyond the coronary tree.

To answer this question, Helgadottir and colleagues compiled clinical and genetic data on the LPA gene from thousands of previous participants in genetic research studies from across the world. They did not have access to Lp(a) levels, but by knowing the genotypes for 2 LPA variants, they inferred the levels of Lp(a) on the basis of prior associations between these variants and Lp(a) levels. [1] Their studies included not only individuals of white European descent but also a significant proportion of black persons, in order to widen the generalizability of their results.

Their main findings are that LPA variants (and, by proxy, Lp(a) levels) are associated with CAD,  peripheral arterial disease, abdominal aortic aneurysm, number of CAD vessels, age at onset of CAD diagnosis, and large-artery atherosclerosis-type stroke. They did not find an association with cardioembolic or small-vessel disease-type stroke; intracranial aneurysm; venous thrombosis; carotid intima thickness; or, in a small subset of individuals, myocardial infarction.

Viewpoint

The main conclusion to draw from this work is that Lp(a) is probably a strong causal factor in not only CAD, but also the development of atherosclerosis in other arterial trees. Although there is no evidence from this study that Lp(a) levels contribute to venous thrombosis, the investigators do not exclude a role for Lp(a) in arterial thrombosis.

Large-artery atherosclerosis stroke is thought to involve some element of arterial thrombosis or thromboembolism, [2] and genetic substudies of randomized trials of aspirin demonstrate that individuals with LPA variants predicted to have elevated levels of Lp(a) benefit the most from antiplatelet therapy. [3] Together, these data suggest that Lp(a) probably has clinically relevant effects on the development of atherosclerosis and arterial thrombosis.

Of  note, the investigators found no association between Lp(a) and carotid intima thickness, suggesting that either intima thickness is a poor surrogate for the clinical manifestations of atherosclerosis or that Lp(a) affects a distinct step in the atherosclerotic disease process that is not demonstrable in the carotid arteries.

Although Lp(a) testing is available, these studies do not provide any evidence that testing for Lp(a) is of clinical benefit, or that screening for atherosclerosis should go beyond well-described clinical risk factors, such as low-density lipoprotein cholesterol levels, high-density lipoprotein levels, hypertension, diabetes, smoking, and family history. Until evidence demonstrates that adding information on Lp(a) levels to routine clinical practice improves the ability of physicians to identify those at highest risk for atherosclerosis, Lp(a) testing should remain a research tool. Nevertheless, these findings do suggest that therapies to lower Lp(a) may have benefits that extend to forms of atherothrombosis beyond the coronary tree.

The finding of this study is interesting:

[1] It consistent with Dr. William LaFramboise..   examination specifically at APO B100, which is part of Lp(a) with some 14 candidate predictors for a more accurate exclusion of patients who don’t need intervention.          Apo B100 was not one of 5 top candidates.

William LaFramboise • Our study (http://www.ncbi.nlm.nih.gov/pubmed/23216991) comprised discovery research using targeted immunochemical screening of retrospective patient samples using both Luminex and Aushon platforms as opposed to shotgun proteomics. Hence the costs constrained sample numbers. Nevertheless, our ability to predict outcome substantially exceeded available methods:

The Framingham CHD scores were statistically different between groups (P <0.001, unpaired Student’s t test) but they classified only 16% of the subjects without significant CAD (10 of 63) at a 95% sensitivity for patients with CAD. In contrast, our algorithm incorporating serum values for OPN, RES, CRP, MMP7 and IFNγ identified 63% of the subjects without significant CAD (40 of 63) at 95% sensitivity for patients with CAD. Thus, our multiplex serum protein classifier correctly identified four times as many patients as the Framingham index.

This study is consistent with the concept of CAD, PVD, and atheromatous disease is a systemic vascular disease, but the point that is made is that it appears to have no relationship to venous thrombosis. The importance for predicting thrombotic events is considered serious.   The venous flow does not have the turbulence of large arteries, so the conclusion is no surprise.  The flow in capillary beds is a linear cell passage with minimal viscosity or turbulence.  The finding of no association with carotid artery disease  is interpreted to mean that the Lp(a) might be an earlier finding than carotid intimal thickness.  It is reassuring to find a recommendation for antiplatelet therapy for individuals with LPA variants based on randomized trials of aspirin substudies.

If that is the conclusion from the studies, and based on the strong association between the prothrombotic (pleiotropic) effect and the association with hyperhomocysteinemia, my own impression is that the recommendation is short-sighted.

[2]  Lp(a) is able to competitively inhibit the binding of plasminogen to fibrinogen and fibrin, and to inhibit the fibrin-dependent activation of plasminogen to plasmin via the tissue plasminogen activator, whereby apo(a) isoforms of low molecular weight have a higher affinity to fibrin than apo(a) isoforms of higher molecular weight. Like other compounds containing sulfhydryl groups, homocysteine enhances the binding of Lp(a) to fibrin.

Prothrombotic :

  • Binding to fibrin
  • Competitive inhibition of plasminogen
  • Stimulation of plasminogen activator inhibitor I and II (PAI -I, PAI -II)
  • Inactivation of tissue factor pathway inhibitor (TFPI)

Source for Lp(a)

Artherogenesis: Predictor of CVD – the Smaller and Denser LDL Particles

https://pharmaceuticalintelligence.com/2012/11/15/artherogenesis-predictor-of-cvd-the-smaller-and-denser-ldl-particles/

References on Triglycerides and blood viscosity

Lowe GD, Lee AJ, Rumley A, et al. Blood viscosity and risk of cardiovascular events: the Edinburgh Artery Study. Br J Haematol 1997; 96:168-173.


Sloop GD. A unifying theory of atherogenesis. Med Hypotheses. 1996; 47:321-5.
Smith WC, Lowe GD, et al. Rheological determinants of blood pressure in a Scottish adult population. J Hypertens 1992; 10:467-72.

Letcher RL, Chien S, et al. Direct relationship between blood pressure and blood viscosity in normal and hypertensive subjects. Role of fibrinogen and concentration. Am J Med 1981; 70:1195-1202.


Devereux RB, Case DB, Alderman MH, et al. Possible role of increased blood viscosity in the hemodynamics of systemic hypertension. Am J Cardiol 2000; 85:1265-1268.


Levenson J, Simon AC, Cambien FA, Beretti C. Cigarette smoking and hypertension. Factors independently associated with blood hyperviscosity and arterial rigidity. Arteriosclerosis 1987; 7:572-577.


Sloop GD, Garber DW. The effects of low-density lipoprotein and high-density lipoprotein on blood viscosity correlate with their association with risk of atherosclerosis in humans. Clin Sci 1997; 92:473-479.

Lowe GD. Blood viscosity, lipoproteins, and cardiovascular risk. Circulation 1992; 85:2329-2331.


Rosenson RS, Shott S, Tangney CC. Hypertriglyceridemia is associated with an elevated blood viscosity: triglycerides and blood viscosity. Atherosclerosis 2002; 161:433-9.


Stamos TD, Rosenson RS. Low high density lipoprotein levels are associated with an elevated blood viscosity. Atherosclerosis 1999; 146:161-5.


Hoieggen A, Fossum E, Moan A, Enger E, Kjeldsen SE. Whole-blood viscosity and the insulin-resistance syndrome. J Hypertens 1998; 16:203-10.


de Simone G, Devereux RB, Chien S, et al. Relation of blood viscosity to demographic and physiologic variables and to cardiovascular risk factors in apparently normal adults. Circulation 1990; 81:107-17.


Rosenson RS, McCormick A, Uretz EF. Distribution of blood viscosity values and biochemical correlates in healthy adults. Clin Chem 1996; 42:1189-95.


Tamariz LJ, Young JH, Pankow JS, et al. Blood viscosity and hematocrit as risk factors for type 2 diabetes mellitus: The Atherosclerosis Risk in Communities (ARIC) Study. Am J Epidemiol 2008; 168:1153-60.


Jax TW, Peters AJ, Plehn G, Schoebel FC. Hemostatic risk factors in patients with coronary artery disease and type 2 diabetes – a two year follow-up of 243 patients. Cardiovasc Diabetol 2009; 8:48.


Ernst E, Weihmayr T, et al. Cardiovascular risk factors and hemorheology. Physical fitness, stress and obesity. Atherosclerosis 1986; 59:263-9.


Hoieggen A, Fossum E, et al. Whole-blood viscosity and the insulin-resistance syndrome. J Hypertens 1998; 16:203-10.


Carroll S, Cooke CB, Butterly RJ. Plasma viscosity, fibrinogen and the metabolic syndrome: effect of obesity and cardiorespiratory fitness. Blood Coagul Fibrinolysis 2000; 11:71-8.


Ernst E, Koenig W, Matrai A, et al. Blood rheology in healthy cigarette smokers. Results from the MONICA project, Augsburg. Arteriosclerosis 1988; 8:385-8.


Ernst E. Haemorheological consequences of chronic cigarette smoking. J Cardiovasc Risk 1995; 2:435-9.


Lowe GD, Drummond MM, Forbes CD, Barbenel JC. The effects of age and cigarette-smoking on blood and plasma viscosity in men. Scott Med J 1980; 25:13-7.


Kameneva MV, Watach MJ, Borovetz HS. Gender difference in rheologic properties of blood and risk of cardiovascular diseases. Clin Hemorheol Microcirc 1999; 21:357-363.


Fowkes FG, Pell JP, Donnan PT, et al. Sex differences in susceptibility to etiologic factors for peripheral atherosclerosis. Importance of plasma fibrinogen and blood viscosity. Arterioscler Thromb 1994; 14:862-8.


Coppola L, Caserta F, De Lucia D, et al. Blood viscosity and aging. Arch Gerontol Geriatr 2000; 31:35-42.

 

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Overview of New Strategy for Treatment of T2DM: SGLT2 Inhibiting Oral Antidiabetic Agents

 

Author and Curator: Aviral Vatsa, PhD, MBBS

Type 2 diabetes mellitus (T2DM) is a chronic disease, which is affecting widespread populations in epidemic proportions across the globe 1. It is characterised by hyperglycemia, which if not controlled adequately, eventually leads to microvascular and metabolic complications (Fig 1). Traditionally, T2DM management includes alteration in lifestyle, oral hypoglycemic agents and/or insulin. The present pharmacological approaches predominantly target glucose metabolism by compensating for reduction in insulin secretion and/or insulin action. However, these approaches are often limited by inadequate glucose control and the the possibility of severe adverse effects such as hypoglycemia, weight gain, nausea, and sometimes lactic acidosis 2–4 (Fig 1). Hence the search for new drugs with different mechanism of action and with little side affects is key in providing better glycemic control in T2DM patients and hence offering better prognosis with reduced morbidity and mortality.

Figure 1 (credit: aviral vatsa): Short overview of Type 2 diabetes mellitus (T2DM): complications, present therapeutic approaches and their limitations.

Along with pancreas, our kidneys play a vital role in regulating glucose levels in the plasma. Under physiological conditions, kidneys absorb 99% of the plasma glucose filtered through the renal glomeruli tubules. Majority i.e. 80-90% of this renal glucose resorbtion is mediated via the sodium glucose co-transporter 2 (SGLT2) 5,6. SGLT2 is a high-capacity low-affinity transporter that is mainly located in the proximal segment S1 of the proximal convoluted tubule 6. Inhibition of SGLT2 activity can thus induce glucosuria which inturn can lower blood glucose levels without targeting insulin resistance and insulin secretion pathways of glucose modulation (Fig 2).

Figure 2 (credit: aviral vatsa): Schematic overview of regulation of plasma glucose by sodium glucose co-transporter (SGLT).

Thus inhibition of SGLT2 provides a novel way to modulate blood glucose levels and consequently limit long term complications of hyperglycemia 7,8. Moreover, SGLT2 inhibitors will selectively target the renal glucose transportation and spare the counter regulatory hormones involved in glucose metabolism because SGLT2 is almost exclusively located in the kidneys. This novel way of glucose modulation will likely avoid severe side affects, e.g. hypoglycemia and weight gain, that are seen with present antidiabetic pharmacological agents.

Agents currently under development

Table below gives an overview of the SGLT2 inhibotors in development.

(Credit: Chao et al 2010)

 

In summary, increasing urinary glucose excretion represents a new approach to addressing the challenge of hyperglycaemia. SGLT2 inhibitors may have indications both in the prevention and treatment of T2DM, and perhaps T1DM, with a possible application in obesity. Further studies in large numbers of human subjects are necessary to delineate efficacy, safety and how to most effectively use these agents in the treatment of diabetes.

Bibliography

  1. Diabetes Atlas. International Diabetes Federation, (2009) at <www.diabetesatlas.org>
  2. Intensive blood-glucose control with sulphonylureas or insulin compared with conventional treatment and risk of complications in patients with type 2 diabetes (UKPDS 33). UK Prospective Diabetes Study (UKPDS) Group. Lancet 352, 837–853 (1998).
  3. Buse, J. B. et al. Effects of exenatide (exendin-4) on glycemic control over 30 weeks in sulfonylurea-treated patients with type 2 diabetes. Diabetes Care 27, 2628–2635 (2004).
  4. Inzucchi, S. E. Oral antihyperglycemic therapy for type 2 diabetes: scientific review. JAMA 287, 360–372 (2002).
  5. Brown, G. K. Glucose transporters: Structure, function and consequences of deficiency. Journal of Inherited Metabolic Disease 23, 237–246 (2000).
  6. Wright, E. M. Renal Na+-glucose cotransporters. Am J Physiol Renal Physiol 280, F10–F18 (2001).
  7. Chao, E. C. & Henry, R. R. SGLT2 inhibition — a novel strategy for diabetes treatment. Nature Reviews Drug Discovery 9, 551–559 (2010).
  8. Ferrannini, E. & Solini, A. SGLT2 inhibition in diabetes mellitus: rationale and clinical prospects. Nature Reviews Endocrinology 8, 495–502 (2012).

 

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