Sergey V. Brodsky

Association of eNOS Glu298Asp Polymorphism With End-Stage Renal Disease

Abstract—Nitric oxide (NO) derived from endothelial cells is profoundly related to the maintenance of physiological vascular tone. Impairment of endothelial NO generation brought about by gene polymorphism is considered the major deterioration factor for progressive renal disease, including diabetic nephropathy. The present study aimed to elucidate the Glu298Asp polymorphism of endothelial NO synthase (eNOS) in patients with end-stage renal disease (ESRD) and its role as a predisposing factor for cardiovascular complications. Glu298Asp in exon 7 of the eNOS gene was determined by polymerase chain reaction, followed by restriction fragment length polymorphism analysis, in ESRD patients (n=185) and compared with that of unrelated healthy individuals (n=304). The occurrence of 298Asp was significantly higher in the ESRD group (P =0.0020; odds ratio [OR] 1.65; 95% confidential interval [CI]: 1.21 to 2.25). In this group, 72 patients had type 2 diabetes mellitus (DM). Although 298Asp did not reach a significant level in the non-DM ESRD subgroup, the occurrence of 298Asp was significantly higher in DM-derived ESRD patients (P =0.0010; OR 2.02; 95% CI: 1.37 to 3.07). The functional effect of the Glu298Asp was examined using Chinese hamster ovary (CHO) cells stably overexpressing either 1917G or 1917T. NO-selective electrode measurements and fluorometric nitrite assay revealed a statistically significant difference in NO production or nitrite accumulation between CHO 1917G and 1917T (P <0.01). These data indicated that Glu298Asp is the predisposing factor in ESRD, especially DM-derived ESRD. The functional difference in NO generation depending on eNOS with either glutamate or aspartate at position 298 was also confirmed in vitro.

Workshop: Endothelial Cell Dysfunction Leading to Diabetic Nephropathy Focus on Nitric Oxide

Clinical manifestations of diabetic nephropathy are an expression of diabetic microangiopathy. This review revisits the previously proposed Steno hypothesis and advances our hypothesis that development of endothelial cell dysfunction represents a common pathophysiological pathway of diabetic complications. Specifically, the ability of glucose to scavenge nitric oxide is proposed as the initiation phase of endothelial dysfunction. Gradual accumulation of advanced glycated end products and induction of plasminogen activator inhibitor-1, resulting in the decreased expression of endothelial nitric oxide synthase and reduced generation of nitric oxide, are proposed to be pathophysiologically critical for the maintenance phase of endothelial dysfunction. The proposed conceptual shift toward the role of endothelial dysfunction in diabetic complications may provide new strategies for their prevention.

Plasminogen Activator Inhibitor-1 Promotes Formation of Endothelial Microparticles With Procoagulant Potential

Background—Endothelial dysfunction is emerging as a common denominator for diverse and highly prevalent cardiovascular diseases. Increased level of plasminogen activator inhibitor-1 (PAI-1) and procoagulant activity have been recognized as hallmarks of endothelial dysfunction. This study was aimed at investigating cellular actions of PAI-1 and a potential link between PAI-1 and procoagulant state. Methods and Results—Human umbilical vein endothelial cells treated with PAI-1 were subjected to laser confocal fluorescence microscopy, immunoprecipitation and Western blotting, and FACS analysis for isolation and identification of endothelial microparticles. PAI-1 treatment resulted in a reduced expression of uPAR, its colocalization with caveolin, and the concomitant increase of uPAR abundance in the culture medium. FACS analysis revealed that PAI-1 rapidly and dose-dependently increased the number of endothelial microparticles expressing uPAR and &agr;V&bgr;3 integrin. This process was attenuated by pretreatment with neutralizing anti-uPAR antibodies. PAI-1 knockout mice showed a significantly decreased number of circulating endothelial microparticles than wild-type mice; however, PAI-1–deficient animals responded to infusion of PAI-1 with a more pronounced rise in the number of microparticles. PAI-1 treatment increased the number of microparticles stained with Annexin V, evidence for the expression of anionic phospholipids. This was accompanied by the accelerated generation of thrombin. Conclusions—The data disclose a novel effect of PAI-1 to dose-dependently promote formation of endothelial microparticles with the reduced transmembrane asymmetry of phospholipids. This phenomenon may be responsible for the observed increase in in vitro thrombin generation. These findings could potentially link these hallmarks of endothelial dysfunction—elevated levels of PAI-1 and propensity toward thrombosis.

Mechanisms of big endothelin-1-induced diuresis and natriuresis : role of ET(B) receptors.

Endothelin-1 (ET-1) at high concentrations has marked antidiuretic and antinatriuretic activities, whereas its precursor, big endothelin-1 (big ET-1), has surprisingly potent diuretic and natriuretic actions. The mechanisms underlying the excretory effects of big ET-1 have not been fully elucidated. To explore these mechanisms, we examined the effects of a highly selective ET(B) antagonist (A-192621.1), a calcium channel blocker (verapamil), a nitric oxide synthase inhibitor (N-nitro-L-arginine methyl ester [L-NAME]), and a cyclooxygenase inhibitor (indomethacin) on the systemic and renal actions of big ET-1 in anesthetized rats. An intravenous bolus injection of incremental doses of big ET-1 (0.3, 1. 0, and 3.0 nmol/kg) produced a significant hypertensive effect that was dose dependent and prolonged (from 113+/-7 mm Hg to a maximum of 148+/-6 mm Hg). The administration of big ET-1 induced marked diuretic and natriuretic responses (urinary flow rate increased from 8.5+/-1 to 110+/-14 microL/min, and fractional excretion of sodium increased from 0.38+/-0.13% to 7.51+/-1.24%). Glomerular filtration rate and renal plasma flow significantly decreased only at the highest dose of big ET-1. Pretreatment with A-192621.1 (3 mg/kg plus 3 mg. kg(-1). h(-1)) significantly abolished the diuretic (17+/-5 microL/min to a maximum of 19+/-3 microL/min) and natriuretic (0. 29+/-0.1% to a maximum of 1.93+/-0.37%) responses induced by big ET-1. Moreover, A-192621.1 potentiated the decline in glomerular filtration rate and renal plasma flow and the increase in mean arterial blood pressure produced by the low doses of big ET-1. Similar to A-192621.1, pretreatment with a nitric oxide synthase inhibitor (L-NAME, 10 mg/kg plus 5 mg. kg(-1). h(-1)) significantly and comparably reduced the diuretic and natriuretic actions of big ET-1 and augmented the hypoperfusion/hypofiltration and systemic vasoconstriction induced by high doses of the peptide. Pretreatment with verapamil (2 mg. kg(-1). h(-1)) slightly inhibited the diuretic/natriuretic effects of the high-dose big ET-1 and completely prevented the increase in mean arterial blood pressure provoked by the peptide. Unlike verapamil and L-NAME, only indomethacin administration was associated with significant natriuretic/diuretic responses and did not influence the pressor effect and renal actions of big ET-1. Taken together, these results suggest that big ET-1-induced diuretic and natriuretic responses are mediated mainly by stimulation of nitric oxide production coupled to ET(B) receptor subtype activation.

Liver sinusoidal endothelial cell modulation upon resection and shear stress in vitro.

BackgroundShear stress forces acting on liver sinusoidal endothelial cells following resection have been noted as a possible trigger in the early stages of hepatic regeneration. Thus, the morphology and gene expression of endothelial cells following partial hepatectomy or shear stress in vitro was studied.ResultsFollowing partial hepatectomy blood flow-to-liver mass ratio reached maximal values 24 hrs post resection. Concomitantly, large fenestrae (gaps) were noted. Exposure of liver sinusoidal endothelial cells, in vitro, to physiological laminar shear stress forces was associated with translocation of vascular endothelial cell growth factor receptor-2 (VEGFR-2) and neuropilin-1 from perinuclear and faint cytoplasmic distribution to plasma membrane and cytoskeletal localization. Under these conditions, VEGFR-2 co-stains with VE-cadherin. Unlike VEGFR-2, the nuclear localization of VEGFR-1 was not affected by shear stress. Quantification of the above receptors showed a significant increase in VEGFR-1, VEGFR-2 and neuropilin-1 mRNA following shear stress.ConclusionOur data suggest a possible relation between elevated blood flow associated with partial hepatectomy and the early events occurring thereby.

Dual Pathways of Carbon Monoxide–Mediated Vasoregulation. Modulation by Redox Mechanisms

Rationale: Vascular tissues produce carbon monoxide (CO) via HO-dependent and HO-independent mechanisms; the former in tandem with biliverdin and iron and the latter as a lone product. CO has been shown to function as both a vasoconstrictor and vasodilator; however, factors that dictate the vasoregulatory phenotype of this gas are unknown. Objective: We investigated whether CO-mediated vasoconstriction is mechanistically linked to enhanced reactive oxygen species production that masks vasodilatory pathways. Methods and Results: Sprague–Dawley rat interlobar and interlobular arteries were examined in terms of superoxide (O2·−) generation and vascular reactivity in the absence and presence of antioxidants. Both authentic CO and the CO-releasing molecule (CORM)-3 constricted renal arteries and increased O2·− production in a dose-dependent manner. The antioxidants tempol, ebselen, and deferoxamine inhibited CO-induced O2·− production and converted CO from constrictor to dilator. CO-induced O2·− generation was found to involve the activity of multiple oxidases including nitric oxide synthase, NADPH oxidase, xanthine oxidase, and complex IV of the mitochondrial electron chain. Furthermore, inhibition of these enzymes converted CO from constrictor to dilator. Similarly, biliverdin and bilirubin inhibited CO-induced O2·− production and vasoconstriction, allowing for a vasodilatory response to CO to be expressed. CO-induced vasoconstriction was dependent on a non-thromboxane agonist of the thromboxane receptor, whereas vasodilatory mechanisms of CO relied on the activation of soluble guanylate cyclase and calcium-gated potassium channels. Conclusions: CO-induced vasoconstriction involves the generation of reactive oxygen species, which, when negated, allows for the expression of vasodilatory pathways which are masked by the primary oxidative stress response to this gas.

Recurrent nested stromal epithelial tumor of the liver with extrahepatic metastasis: case report and review of literature.

Nested stromal epithelial tumor is a recently described primary neoplasm of the liver. This tumor is characterized by well-demarcated nests of spindle and epithelioid cells with occasional calcification and bone formation. An association between these tumors and Cushing syndrome has been described. Herein we report a case of a recurrent nested stromal epithelial tumor of the liver in a 17-year-old female with aggressive clinical behavior and an extrahepatic lymph node metastasis. Also, we provide the first detailed clinical, histologic, immunohistochemical, and cytogenetic comparison of the original and recurrent tumors. Initially, the patient presented with Cushingoid symptoms and epigastric pain, radiating to her back. A computed tomographic (CT) scan revealed a large lesion in the liver. After a partial hepatectomy, the Cushingoid features were resolved. A year later, a CT scan revealed multiple lesions within the liver, and positron emission tomographic/CT imaging showed a hypermetabolic lymph node. The patient underwent a cadaveric liver transplant. Histologically, both the original and recurrent tumors had similar characteristics, with different immunoreactivity, correlating with the absence of systemic hormonal symptoms. Electron microscopy of the original neoplasm revealed an abundance of rough cytoplasmic reticulum and mitochondria. No evidence of endocrine differentiation was found. Cytogenetics of the primary tumor was complex with an abnormal hypotriploid karyotype. Our data indicate that patients with nested stromal epithelial tumor of the liver must be carefully followed with imaging to detect hepatic recurrence and extrahepatic metastases.

Coagulation, fibrinolysis and angiogenesis: new insights from knockout mice.

Angiogenesis plays a key role in a broad array of physiologic and pathologic processes. Two major systems – coagulation and fibrinolysis – maintaining hemostasis, have recently been implicated in angiogenesis. Generation of mice deficient in components of coagulation and plasminogen systems has provided an extraordinary opportunity to define the role of each of these systems in vivo and to elucidate molecular mechanisms involved in angiogenesis. It appears that several factors of the coagulation system, such as the tissue factor, the factor V and the thrombin receptor, play an important role in embryonic vessel formation, most probably in the formation of the primitive vascular wall. In addition, the plasminogen system appears to play a significant role in angiogenesis in adulthood, regulating the migration of endothelial and smooth muscle cells, the degradation of the extracellular matrix and activity of the metalloproteinase system. These new revelations open a possibility for future therapeutic strategies to specifically control angiogenesis in different pathological processes where abnormalities of tissue vascularization are pathogenetically prominent.

Effects of A-192621.1, a specific endothelin-B antagonist, on intrarenal hemodynamic responses to endothelin-1

The present study examined the effects of A-192621.1, a highly selective endothelin-B- (ETB) receptor antagonist, on the renal hemodynamic and systemic actions of endothelin-1 (ET-1). Intravenous injection of ET-1 (1.0 nmol/kg) into anesthetized rats produced a sustained decrease in renal blood flow (assessed by ultrasonic flowmeter) and a significant increase in renal vascular resistance, as well as an increase in mean arterial pressure. These changes were significantly augmented by pretreatment with A-192621.1 (3.0 mg/kg/h). Analysis of intrarenal blood flow by laser-Doppler flowmeter revealed that ET-1 caused a marked and sustained decrease in cortical blood flow, associated with a transient increase in medullary blood flow. The reduction in cortical blood flow in response to ET-1 was further enhanced by pretreatment with A-192621.1, whereas the ET-1-induced medullary vasodilatation was completely abolished and reversed into a vasoconstrictor response. These findings suggest that the ETB-receptors mediate the systemic and renal vasodilatory actions of ET-1 in the rat, and that their activation may serve as a physiological counterbalance that modulates ET-1-induced vasoconstriction.

A model for ex vivo renal angiogenesis.

Attempts to study renal angiogenesis have been hampered by the lack of an appropriate model. Here we present data on a successful ex vivo culture of renal medullary explants in three-dimensional collagen 1 or Matrigel lattices and characterize the dynamics of capillary formation by sprouting endothelial cells. Initially, endothelial cells represented 71 ± 3% among the sprouting cells, but within a week growing capillaries were comprised exclusively of endothelial cells. The quantitative analysis showed that the number of sprouting capillaries progressively increased until 12 days in culture, after which capillaries underwent involution. Occasional formation of glomeruloid bodies was noted. Capillaries were characterized by a well-defined lumen, whereas glomeruloid bodies showed cellular debris occupying the luminal space. In view of the existing controversy regarding angiogenic competence in diabetic nephropathy, we applied this ex vivo culture system to Zucker diabetic rat model of diabetes mellitus. Comparative analysis of capillary sprouting in Zucker diabetic fat and lean nondiabetic control rats showed no differences in angiogenic properties of renal explants obtained at the age of 11 weeks. However, when kidneys were obtained from rats at age of 21 weeks, the capillary sprouting was significantly reduced in Zucker diabetic rats compared to age-matched lean rats. The rate of capillary involution was unaffected in Zucker diabetic rats. In conclusion, the data presented herein delineate the first successful ex vivo model of angiogenesis initiated from the renal medullary explants of adult rats and provide evidence of impaired angiogenesis in Zucker diabetic rats with the established, but not with incipient diabetes mellitus.