Domokos Gero

Hydrogen sulfide and nitric oxide are mutually dependent in the regulation of angiogenesis and endothelium-dependent vasorelaxation

Hydrogen sulfide (H2S) is a unique gasotransmitter, with regulatory roles in the cardiovascular, nervous, and immune systems. Some of the vascular actions of H2S (stimulation of angiogenesis, relaxation of vascular smooth muscle) resemble those of nitric oxide (NO). Although it was generally assumed that H2S and NO exert their effects via separate pathways, the results of the current study show that H2S and NO are mutually required to elicit angiogenesis and vasodilatation. Exposure of endothelial cells to H2S increases intracellular cyclic guanosine 5′-monophosphate (cGMP) in a NO-dependent manner, and activated protein kinase G (PKG) and its downstream effector, the vasodilator-stimulated phosphoprotein (VASP). Inhibition of endothelial isoform of NO synthase (eNOS) or PKG-I abolishes the H2S-stimulated angiogenic response, and attenuated H2S-stimulated vasorelaxation, demonstrating the requirement of NO in vascular H2S signaling. Conversely, silencing of the H2S-producing enzyme cystathionine-γ-lyase abolishes NO-stimulated cGMP accumulation and angiogenesis and attenuates the acetylcholine-induced vasorelaxation, indicating a partial requirement of H2S in the vascular activity of NO. The actions of H2S and NO converge at cGMP; though H2S does not directly activate soluble guanylyl cyclase, it maintains a tonic inhibitory effect on PDE5, thereby delaying the degradation of cGMP. H2S also activates PI3K/Akt, and increases eNOS phosphorylation at its activating site S1177. The cooperative action of the two gasotransmitters on increasing and maintaining intracellular cGMP is essential for PKG activation and angiogenesis and vasorelaxation. H2S-induced wound healing and microvessel growth in matrigel plugs is suppressed by pharmacological inhibition or genetic ablation of eNOS. Thus, NO and H2S are mutually required for the physiological control of vascular function.

Hydrogen sulfide replacement therapy protects the vascular endothelium in hyperglycemia by preserving mitochondrial function

The goal of the present studies was to investigate the role of changes in hydrogen sulfide (H2S) homeostasis in the pathogenesis of hyperglycemic endothelial dysfunction. Exposure of bEnd3 microvascular endothelial cells to elevated extracellular glucose (in vitro “hyperglycemia”) induced the mitochondrial formation of reactive oxygen species (ROS), which resulted in an increased consumption of endogenous and exogenous H2S. Replacement of H2S or overexpression of the H2S-producing enzyme cystathionine-γ-lyase (CSE) attenuated the hyperglycemia-induced enhancement of ROS formation, attenuated nuclear DNA injury, reduced the activation of the nuclear enzyme poly(ADP-ribose) polymerase, and improved cellular viability. In vitro hyperglycemia resulted in a switch from oxidative phosphorylation to glycolysis, an effect that was partially corrected by H2S supplementation. Exposure of isolated vascular rings to high glucose in vitro induced an impairment of endothelium-dependent relaxations, which was prevented by CSE overexpression or H2S supplementation. siRNA silencing of CSE exacerbated ROS production in hyperglycemic endothelial cells. Vascular rings from CSE−/− mice exhibited an accelerated impairment of endothelium-dependent relaxations in response to in vitro hyperglycemia, compared with wild-type controls. Streptozotocin-induced diabetes in rats resulted in a decrease in the circulating level of H2S; replacement of H2S protected from the development of endothelial dysfunction ex vivo. In conclusion, endogenously produced H2S protects against the development of hyperglycemia-induced endothelial dysfunction. We hypothesize that, in hyperglycemic endothelial cells, mitochondrial ROS production and increased H2S catabolism form a positive feed-forward cycle. H2S replacement protects against these alterations, resulting in reduced ROS formation, improved endothelial metabolic state, and maintenance of normal endothelial function.

MD-2 is required for disulfide HMGB1–dependent TLR4 signaling

Yang et al. show that a disulfide isoform of HMGB1, with a role in TLR4 signaling, physically interacts with and binds MD-2. MD-2 deficiency in macrophage cell lines or in primary mouse macrophages stimulated with HMGB1 implicates MD-2 in TLR4 signaling. They also identify an HGMB1 peptide inhibitor, P5779, which when administered in vivo can protect mice from acetaminophen-induced hepatoxicity, ischemia/reperfusion injury, and sepsis.

The peroxynitrite decomposition catalyst FP15 improves ageing-associated cardiac and vascular dysfunction

Overproduction of oxidants and free radicals in ageing tissues induces nitro-oxidative stress, which has recently been implicated in the pathogenesis of cardiovascular dysfunction associated with ageing. Peroxynitrite, a strong cytotoxic oxidant damages proteins and DNA and activates several pathways causing tissue injury, including the peroxynitrite-poly(ADP-ribose) polymerase (PARP) pathway. In this study, we investigated the effectiveness of the peroxynitrite decomposition catalyst FP15 on ageing-associated cardiac and vascular dysfunction. Young and ageing rats were treated with vehicle or FP15 intraperitoneally. Using a microtip Millar pressure catheter we performed left ventricular blood pressure analysis to assess systolic and diastolic function. Endothelium-dependent and -independent vasorelaxation of isolated aortic rings were investigated by using acetylcholine and sodium nitroprusside. Ageing animals showed a marked reduction of systolic and diastolic cardiac function and loss of endothelium-dependent relaxant responsiveness of aortic rings. FP15-treatment significantly improved cardiac performance and endothelial function. Immunohistochemical staining confirmed that FP15 effectively reduced nitrosative stress and prevented the activation of PARP in the aortic wall of ageing rats. Our results demonstrate the importance of endogenous peroxynitrite-overproduction in the pathogenesis of ageing-associated cardiovascular dysfunction. Pharmacological decomposition of peroxynitrite by FP15 may represent a novel therapeutic utility to improve cardiac and vascular dysfunction associated with ageing.

Poly(ADP-ribose) polymerase: a new therapeutic target?

Purpose of review To overview the emerging data in the literature showing the role of poly(ADP-ribose) polymerase (PARP) in the pathogenesis of critical illness. Recent findings PARP, an abundant nuclear enzyme involved in DNA repair and transcriptional regulation, is now recognized as a key regulator of cell survival and cell death in response to noxious stimuli in various forms of cardiovascular collapse. PARP becomes activated in response to oxidative DNA damage and depletes cellular energy pools, thus leading to cellular dysfunction in various tissues. The activation of PARP may also induce various cell death processes, and promotes an inflammatory response. In circulatory shock PARP plays a crucial role both in the development of early cardiovascular dysfunction and in the delayed systemic inflammatory response syndrome with associated multiple organ failure. Inhibition of PARP activity is protective in various models of circulatory shock. Summary A solid body of literature supports the view that PARP is an important target for therapeutic intervention in critical illness.

Single dose treatment with PARP-inhibitor INO-1001 improves aging-associated cardiac and vascular dysfunction.

Overproduction of reactive oxygen species in aging tissues has been implicated in the pathogenesis of aging-associated cardiovascular dysfunction. Oxidant-induced DNA-damage activates the poly(ADP-ribose) polymerase (PARP) pathway, leading to tissue injury. In this study we investigated the acute effects of the PARP inhibitor INO-1001 on aging-associated cardiac and endothelial dysfunction. Using a pressure-volume conductance catheter, left ventricular pressure-volume analysis of young and aging rats was performed before and after a single injection of INO-1001. Endothelium-dependent and -independent vasorelaxation of isolated aortic rings were investigated by using acetylcholine and sodium nitroprusside. Aging animals showed a marked reduction of myocardial contractility and endothelium-dependent relaxant responsiveness of aortic rings. Single dose INO-1001-treatment resulted in acute improvement in their cardiac and endothelial function. Immunohistochemistry for nitrotyrosine and poly(ADP-ribose) confirmed enhanced nitro-oxidative stress and PARP-activation in aging animals. Acute treatment with INO-1001 decreased PARP-activation, but did not affect nitrotyrosine-immunoreactivity. Our results demonstrate that the aging-associated chronic cardiovascular dysfunction can be improved, at least, short term, by a single treatment course with a PARP-inhibitor, supporting the role of the nitro-oxidative stress -- PARP -- pathway in the age-related functional decline of the cardiovascular system. Pharmacological inhibition of PARP may represent a novel therapeutic utility to improve aging-associated cardiovascular dysfunction.

A cell-microelectronic sensing technique for the screening of cytoprotective compounds.

In recent years, a new cell-based high throughput paradigm has emerged, which seeks to identify novel, pharmacologically active cytoprotective compounds. The essence of this approach is to create experimental models of cell injury relevant for a particular disease by establishing in vitro cell-based models, followed by high-throughput testing of compounds that affect the cellular response in a desired manner. Prior approaches typically used simple end-point analyses. To assess the cytoprotective effects of novel drug candidates in real-time, we have applied a cell-microelectronic sensing technique (RT-CES), which measures changes in the impedance of individual microelectronic wells that correlates linearly with cell index (reflecting cell number, adherence and cell growth), thereby allowing the continuous determination of cell viability during oxidative stress. In vitro cytotoxicity was elicited by hydrogen peroxide in myocytes (H9c2) and hepatocytes (Hep3B). Cells were post-treated at 30 min with various reference molecules and novel cytoprotective compounds. Cytoprotection detected in the RT-CES system correlated well with the results of two classical end-point-based methods (improvement in MTT and reduction of LDH release). The RT-CES method, when used as described in the current report, is suitable for the screening of molecular libraries to identify molecules or molecule combinations that attenuate oxidative stress-induced cell damage.

Cytoprotective effects of adenosine and inosine in an in vitro model of acute tubular necrosis

Background and purpose:  We have established an in vitro model of acute tubular necrosis in rat kidney tubular cells, using combined oxygen‐glucose deprivation (COGD) and screened a library of 1280 pharmacologically active compounds for cytoprotective effects.

Cell-Based Screening Identifies Paroxetine as an Inhibitor of Diabetic Endothelial Dysfunction

We have conducted a phenotypic screening in endothelial cells exposed to elevated extracellular glucose (an in vitro model of hyperglycemia) to identify compounds that prevent hyperglycemia-induced reactive oxygen species (ROS) formation without adversely affecting cell viability. From a focused library of >6,000 clinically used drug-like and pharmacologically active compounds, several classes of active compounds emerged, with a confirmed hit rate of <0.5%. Follow-up studies focused on paroxetine, a clinically used antidepressant compound that has not been previously implicated in the context of hyperglycemia or diabetes. Paroxetine reduced hyperglycemia-induced mitochondrial ROS formation, mitochondrial protein oxidation, and mitochondrial and nuclear DNA damage, without interfering with mitochondrial electron transport or cellular bioenergetics. The ability of paroxetine to improve hyperglycemic endothelial cell injury was unique among serotonin reuptake blockers and can be attributed to its antioxidant effect, which primarily resides within its sesamol moiety. Paroxetine maintained the ability of vascular rings to respond to the endothelium-dependent relaxant acetylcholine, both during in vitro hyperglycemia and ex vivo, in a rat model of streptozotocin-induced diabetes. Thus, the current work identifies a novel pharmacological action of paroxetine as a protector of endothelial cells against hyperglycemic injury and raises the potential of repurposing of this drug for the experimental therapy of diabetic cardiovascular complications.

Poly(ADP-Ribose) Polymerase Inhibition Improves Endothelial Dysfunction Induced by Hypochlorite

Reactive oxygen species, such as myeloperoxidase-derived hypochlorite, induce oxidative stress and DNA injury. The subsequent activation of the DNA-damage–poly(ADP-ribose) polymerase (PARP) pathway has been implicated in the pathogenesis of various diseases, including ischemia-reperfusion injury, circulatory shock, diabetic complications, and atherosclerosis. We investigated the effect of PARP inhibition on the impaired endothelium-dependent vasorelaxation induced by hypochlorite. In organ bath experiments for isometric tension, we investigated the endothelium-dependent and endothelium-independent vasorelaxation of isolated rat aortic rings using cumulative concentrations of acetylcholine and sodium nitro-prusside. Endothelial dysfunction was induced by exposing rings to hypochlorite (100–400 μM). In the treatment group, rings were preincubated with the PARP inhibitor INO-1001. DNA strand breaks were assessed by the TUNEL method. Immunohistochemistry was performed for 4-hydroxynonenal (a marker of lipid peroxidation), nitrotyrosine (a marker of nitrosative stress), and poly(ADP-ribose) (an enzymatic product of PARP). Exposure to hypochlorite resulted in a dose-dependent impairment of endothelium-dependent vasorelaxation of aortic rings, which was significantly improved by PARP inhibition, whereas the endothelium-independent vasorelaxation remained unaffected. In the hypochlorite groups we found increased DNA breakage, lipidperoxidation, and enhanced nitrotyrosine formation. The hypochloride-induced activation of PARP was prevented by INO-1001. Our results demonstrate that PARP activation contributes to the pathogenesis of hypochlorite-induced endothelial dysfunction, which can be prevented by PARP inhibitors.