Sourav Kundu

Platelet GP IIIa Pl(A) polymorphisms display different sensitivities to agonists

BACKGROUND Both inherited predisposition and platelet hyperreactivity have been associated with ischemic coronary events, but mechanisms that support genetic differences among platelets from different subjects are generally lacking. Associations between the platelet Pl(A2) polymorphism of GP IIIa and coronary syndromes raise the question as to whether this inherited variation may contribute to platelet hyperreactivity. METHODS AND RESULTS In this study, we characterized functional parameters in platelets from healthy donors with the Pl(A) (HPA-1) polymorphism, a Leu (Pl(A1)) to Pro (Pl(A2)) substitution at position 33 of the GP IIIa subunit of the platelet GP IIb/IIIa receptor (integrin alpha(IIb)beta(3)). We studied 56 normal donors (20 Pl(A1,A1), 20 Pl(A1,A2), and 16 Pl(A2,A2)). Compared with Pl(A1,A1) platelets, Pl(A2)-positive platelets showed a gene dosage effect for significantly greater surface-expressed P-selectin, GP IIb/IIIa-bound fibrinogen, and activated GP IIb/IIIa in response to low-dose ADP. Surface expression of GP IIb/IIIa was similar in resting platelets of all 3 genotypes but was significantly greater on Pl(A2,A2) platelets after ADP stimulation (P=0.003 versus Pl(A1,A1); P=0.03 versus Pl(A1,A2)). Pl(A1,A2) platelets were more sensitive to inhibition of aggregation by pharmacologically relevant concentrations of aspirin and abciximab. CONCLUSIONS Pl(A2)-positive platelets displayed a lower threshold for activation, and platelets heterozygous for Pl(A) alleles showed increased sensitivity to 2 antiplatelet drugs. These in vitro platelet studies may have relevance for in vivo thrombotic conditions.

The effect of antiplatelet drugs, heparin, and preanalytical variables on platelet function detected by the platelet function analyzer (PFA-100).

The platelet function analyzer (PFA)-100® is a newly developed instrument that provides a rapid, in vitro, quantitative measurement of platelet adhesion and aggregation in whole blood flowing through a small aperture under high shear conditions. Thirty patients undergoing percutaneous transluminal coronary angioplasty (PTCA) and ten normal in dividuals were included in this study. In vitro and in vivo studies were conducted to discern the effect of combinations of antiplatelet drugs (aspirin, ticlopidine, abciximab) and heparin on the performance of the device as well as the effects of preanalytical variables, such as method of sample collection and ex vivo anticoagulants. Studies were also conducted ex amining the effect of aperture size (standard 150 μm vs. smaller 120 μm) on the ability of the device to detect the effect of antiplatelet drugs. There was no difference in mean PFA- 100® closure time with citrate versus PPACK anticoagulants or with venipuncture vs. sheath sampling. Closure times did not vary with heparin administration. Closure times were slightly longer for patients taking aspirin plus ticlopidine compared to aspirin alone (p = NS). In contrast adenosine disphosphate (ADP) induced platelet aggregation was significantly less in patients that took aspirin plus ticlopidine vs. aspirin alone (p = .0005). In vitro, there was a dose-dependent increase in closure time for both aperture sizes with increasing abciximab concen tration. Although both cartridges showed infinite closure times at an abciximab concentration of 2.25 μg/mL, there was a slight benefit to using the 120 μm aperture cartridges at abcix imab concentrations of 1.75 to 2.0 μg/mL. In ten patients who were followed during abciximab therapy to assess the effect of aperture size, the PFA-100® was able to detect in vivo platelet inhibition by abciximab, but detection of recovery from abcix imab-induced platelet dysfunction was slightly better for the PFA-100® with the 120 μm aperture compared to the standard 150 μm aperture collagen/ADP cartridge.

COMPARISON OF FOUR COMMERCIAL CITRATE BLOOD COLLECTION SYSTEMS FOR PLATELET FUNCTION ANALYSIS BY THE PFA-100TM SYSTEM1.

The PFA-l OOTMsystem is a recently developed in vitro test system for measurement of platelet fi.mction in whole blood anticoagulated with sodium citrate. The test system is based on the principle originally described by Kratzer and Born (l), and has been characterized elsewhere (2,3). In this system, a motor-driven syringe aspirates blood samples under steady-flow conditions through a small aperture (approximately 150 pm in diameter) cut into a membrane placed in the test cartridge. The membrane is coated with type I collagen and either epinephrine (Col/Epi) or adenosine-5’-diphosphate (Col/ADP). Progressively, platelets attach to the area surrounding the aperture and eventually form a platelet plug that occludes it. The instrument records the time necessary for the occlusion of the aperture, defined as the Closure Time (CT), which is indicative of platelet function in the blood sample.

Increased endogenous H2S generation by CBS, CSE, and 3MST gene therapy improves ex vivo renovascular relaxation in hyperhomocysteinemia

Hydrogen sulfide (H(2)S) has recently been identified as a regulator of various physiological events, including vasodilation, angiogenesis, antiapoptotic, and cellular signaling. Endogenously, H(2)S is produced as a metabolite of homocysteine (Hcy) by cystathionine β-synthase (CBS), cystathionine γ-lyase (CSE), and 3-mercaptopyruvate sulfurtransferase (3MST). Although Hcy is recognized as vascular risk factor at an elevated level [hyperhomocysteinemia (HHcy)] and contributes to vascular injury leading to renovascular dysfunction, the exact mechanism is unclear. The goal of the current study was to investigate whether conversion of Hcy to H(2)S improves renovascular function. Ex vivo renal artery culture with CBS, CSE, and 3MST triple gene therapy generated more H(2)S in the presence of Hcy, and these arteries were more responsive to endothelial-dependent vasodilation compared with nontransfected arteries treated with high Hcy. Cross section of triple gene-delivered renal arteries immunostaining suggested increased expression of CD31 and VEGF and diminished expression of the antiangiogenic factor endostatin. In vitro endothelial cell culture demonstrated increased mitophagy during high levels of Hcy and was mitigated by triple gene delivery. Also, dephosphorylated Akt and phosphorylated FoxO3 in HHcy were reversed by H(2)S or triple gene delivery. Upregulated matrix metalloproteinases-13 and downregulated tissue inhibitor of metalloproteinase-1 in HHcy were normalized by overexpression of triple genes. Together, these results suggest that H(2)S plays a key role in renovasculopathy during HHcy and is mediated through Akt/FoxO3 pathways. We conclude that conversion of Hcy to H(2)S by CBS, CSE, or 3MST triple gene therapy improves renovascular function in HHcy.

Endothelial Dysfunction: The Link Between Homocysteine and Hydrogen Sulfide

High level of homocysteine (hyperhomocysteinemia, HHcy) is associated with increased risk for vascular disease. Evidence for this emerges from epidemiological studies which show that HHcy is associated with premature peripheral, coronary artery and cerebrovascular disease independent of other risk factors. Possible mechanisms by which homocysteine causes vascular injury include endothelial injury, DNA dysfunction, proliferation of smooth muscle cells, increased oxidative stress, reduced activity of glutathione peroxidase and promoting inflammation. HHcy has been shown to cause direct damage to endothelial cells both in vitro and in vivo. Clinically, this manifests as impaired flow-mediated vasodilation and is mainly due to a reduction in nitric oxide synthesis and bioavailability. The effect of impaired nitric oxide release can in turn trigger and potentiate atherothrombogenesis and oxidative stress. Endothelial damage is a crucial aspect of atherosclerosis and precedes overt manifestation of disease. In addition, endothelial dysfunction is also associated with hypertension, diabetes, ischemia reperfusion injury and neurodegenerative diseases. Homocysteine is a precursor of hydrogen sulfide (H2S) which is formed by transulfuration process catalyzed by the enzymes, cystathionine β-synthase and cystathionine γ-lyase. H2S is a gasotransmitter that has emerged recently as a novel mediator in cardiovascular homeostasis. As a potent vasodilator, it plays several roles which include regulation of vessel diameter, protection of endothelium from redox stress, ischemia reperfusion injury and chronic inflammation. However, the precise mechanism by which it mediates these beneficial effects is complex and still remains unclear. Current evidence indicates H2S modulates cellular functions by a variety of intracellular signaling processes. In this review, we summarize the mechanisms of HHcy-induced endothelial dysfunction and the metabolism and physiological functions of H2S as a protective agent.

Regulation of ovarian steroidogenesis in vitro by gonadotropin in common carp Cyprinus carpio: interaction between calcium- and adenylate cyclase-dependent pathways and involvement of ERK signaling cascade

Multiple signal transduction pathways mediating gonadotropin-induced testosterone and 17β-estradiol (E(2)) production were identified in carp ovarian theca and granulosa cells in short-term co-incubation. Inhibitors of voltage-sensitive calcium channels (VSCCs) and calmodulin attenuated human chorionic gonadotropin (HCG)-induced steroid production, whereas modulators of adenylate cyclase and protein kinase A (PKA) increased their production, indicating that both calcium- and PKA-dependent pathways are involved in the regulation of gonadotropin-induced steroidogenesis in carp ovary. Interactions between these two pathways are evident from the positive effect of elevated intracellular calcium on HCG-induced steroid production and the reduction of forskolin (FK)- and dibutyryl cAMP (dbcAMP)-induced steroidogenesis by inhibitors of VSCCs and calmodulin. In this study, we found the involvement of a third signaling pathway, a mitogen-activated protein kinase (MAP kinase), in the regulation of gonadal steroidogenesis in this fish. An antagonist of mitogen-activated protein kinase kinases 1/2 (MEK1/2; also known as MAP2K1/MAP2K2) markedly attenuated HCG-induced steroid production. Cells treated with HCG stimulated MEK1/2-dependent phosphorylation of extracellular signal-regulated protein kinases 1/2 (ERKs1/2) in a concentration and time-dependent manner. Moreover, ERK1/2 activation in cells was mimicked by FK and dbcAMP suggesting that ERK1/2 transduce signal downstream of PKA in HCG-induced ovarian steroidogenesis. Evidence for presence of cross talk between calcium-dependent pathways and this MAP kinase cascade has been shown by demonstrating the inhibitory effects of verapamil and calmodulin on ERK1/2 activation after HCG stimulation. Our results suggest that activation of ERK1/2 by HCG as well as other agents may be a key mechanism for the modulation of gonadotropin-induced steroidogenesis in carp ovary.

Stimulation of salmon calcitonin on secretion of 17β-estradiol by the ovarian follicles of common carp, Cyprinus carpio

The effects of salmon calcitonin (sCT) on the secretion of 17beta-estradiol (E(2)) were examined in female common carp, Cyprinus carpio. Vitellogenic stage fish adapted to high-Ca water were i.p. injected with vehicle, sCT, human chorionic gonadotropin (hCG), or hCG plus sCT. To determine whether ovarian follicles are equipped with CT receptors, a CT binding assay was conducted. In the in vitro experiments, vitellogenic follicles were incubated with stimulators and inhibitors. Administration of sCT increased the basal and hCG-stimulated E(2) release in vivo and in vitro. Binding characteristics of [(125)I]sCT to plasma membrane preparation of carp ovarian follicles showed saturability with high-affinity (K(d)=48.48 pmol/l and B(max)=1.2 pmol/mg protein). To clarify the mechanism of E(2) production by sCT, in vitro effect of sCT and hCG on aromatase activity (conversion of testosterone to E(2)) and cytochrome P450 aromatase (P450arom) gene expression in carp ovarian follicles were investigated. Salmon CT-stimulated both aromatase activity and P450arom gene expression in ovarian follicles of carp. sCT-stimulated E(2) release by the ovarian follicles in vitro was augmented in the presence of dibutyryl cAMP. Inhibitor of protein kinase A (PKA), SQ 22536 inhibited sCT-stimulated steroid production in a dose-dependent manner. Specific inhibitor of protein kinase C (PKC), NPC-15437 dihydrochloride had no inhibitory effects on sCT-induced E(2) release. The present study indicates that sCT binds specifically to carp ovary and stimulates E(2) production by increasing the activity of cytochrome P450 aromatase and P450arom gene expression. The results further suggest that stimulatory action of sCT on E(2) production is mediated through cAMP pathway.

Moderate intensity exercise prevents diabetic cardiomyopathy associated contractile dysfunction through restoration of mitochondrial function and connexin 43 levels in db/db mice

AIMS Although the cardiovascular benefits of exercise are well known, exercise induced effects and mechanisms in prevention of cardiomyopathy are less clear during obesity associated type-2 diabetes. The current study assessed the impact of moderate intensity exercise on diabetic cardiomyopathy by examining cardiac function and structure and mitochondrial function. METHODS Obese-diabetic (db/db), and lean control (db/+) mice, were subjected to a 5 week, 300 m run on a tread-mill for 5 days/week at the speeds of 10-11 m/min. Various physiological parameters were recorded and the heart function was evaluated with M-mode echocardiography. Contraction parameters and calcium transits were examined on isolated cardiomyocytes. At the molecular level: connexin 43 and 37 (Cx43 and 37) levels, mitochondrial biogenesis regulators: Mfn2 and Drp-1 levels, mitochondrial trans-membrane potential and cytochrome c leakage were assessed through western blotting immunohistochemistry and flow cytometry. Ability of exercise to reverse oxygen consumption rate (OCR), tissue ATP levels, and cardiac fibrosis were also determined. RESULTS The exercise regimen was able to prevent diabetic cardiac functional deficiencies: ejection fraction (EF) and fractional shortening (FS). Improvements in contraction velocity and contraction maximum were noted with the isolated cardiomyocytes. Restoration of interstitial and micro-vessels associated Cx43 levels and improved gap junction intercellular communication (GJIC) were observed. The decline in the Mfn2/Drp-1 ratio in the db/db mice hearts was prevented after exercise. The exercise regimen further attenuated transmembrane potential decline and cytochrome c leakage. These corrections further led to improvements in OCR and tissue ATP levels and reduction in cardiac fibrosis. CONCLUSIONS Moderate intensity exercise produced significant cardiovascular benefits by improving mitochondrial function through restoration of Cx43 networks and mitochondrial trans-membrane potential and prevention of excessive mitochondrial fission.

Hydrogen sulfide mitigates hyperglycemic remodeling via liver kinase B1-adenosine monophosphate-activated protein kinase signaling

Hyperglycemia (HG) reduces AMPK activation leading to impaired autophagy and matrix accumulation. Hydrogen sulfide (H2S) treatment improves HG-induced renovascular remodeling however, its mechanism remains unclear. Activation of LKB1 by the formation of heterotrimeric complex with STRAD and MO25 is known to activate AMPK. We hypothesized that in HG; H2S induces autophagy and modulates matrix synthesis through AMPK-dependent LKB1/STRAD/MO25 complex formation. To address this hypothesis, mouse glomerular endothelial cells were treated with normal and high glucose in the absence or presence of sodium hydrogen sulfide (NaHS), an H2S donor. HG decreased the expression of H2S regulating enzymes CBS and CSE, and autophagy markers Atg5, Atg7, Atg3 and LC3B/A ratio. HG increased galectin-3 and periostin, markers of matrix accumulation. Treatment with NaHS to HG cells increased LKB1/STRAD/MO25 formation and AMPK phosphorylation. Silencing the encoded genes confirmed complex formation under normoglycemia. H2S-mediated AMPK activation in HG was associated with upregulation of autophagy and diminished matrix accumulation. We conclude that H2S mitigates adverse remodeling in HG by induction of autophagy and regulation of matrix metabolism through LKB1/STRAD/MO25 dependent pathway.

Folic Acid Mitigates Angiotensin-II-Induced Blood Pressure and Renal Remodeling

Clinical data suggests an association between systolic hypertension, renal function and hyperhomocysteinemia (HHcy). HHcy is a state of elevated plasma homocysteine (Hcy) levels and is known to cause vascular complications. In this study, we tested the hypothesis whether Ang II-induced hypertension increases plasma Hcy levels and contributes to renovascular remodeling. We also tested whether folic acid (FA) treatment reduces plasma Hcy levels by enhancing Hcy remethylation and thus mitigating renal remodeling. Hypertension was induced in WT mice by infusing Ang II using Alzet mini osmotic pumps. Blood pressure, Hcy level, renal vascular density, oxidative stress, inflammation and fibrosis markers, and angiogenic- and anti-angiogenic factors were measured. Ang II hypertension increased plasma Hcy levels and reduced renal cortical blood flow and microvascular density. Elevated Hcy in Ang II hypertension was associated with decreased 4, 5-Diaminofluorescein (DAF-2DA) staining suggesting impaired endothelial function. Increased expression of Nox-2, -4 and dihydroethidium stain revealed oxidative stress. Excess collagen IV deposition in the peri-glomerular area and increased MMP-2, and -9 expression and activity indicated renal remodeling. The mRNA and protein expression of asymmetric dimethylarginine (ADMA) was increased and eNOS protein was decreased suggesting the involvement of this pathway in Hcy mediated hypertension. Decreased expressions of VEGF and increased anti-angiogenic factors, angiostatin and endostatin indicated impaired vasculogenesis. FA treatment partially reduced hypertension by mitigating HHcy in Ang II-treated animals and alleviated pro-inflammatory, pro-fibrotic and anti-angiogenic factors. These results suggest that renovascular remodeling in Ang II-induced hypertension is, in part, due to HHcy.