David Lominadze

Mechanisms of fibrinogen‐induced microvascular dysfunction during cardiovascular disease

Fibrinogen (Fg) is a high molecular weight plasma adhesion protein and a biomarker of inflammation. Many cardiovascular and cerebrovascular disorders are accompanied by increased blood content of Fg. Increased levels of Fg result in changes in blood rheological properties such as increases in plasma viscosity, erythrocyte aggregation, platelet thrombogenesis, alterations in vascular reactivity and compromises in endothelial layer integrity. These alterations exacerbate the complications in peripheral blood circulation during cardiovascular diseases such as hypertension, diabetes and stroke. In addition to affecting blood viscosity by altering plasma viscosity and erythrocyte aggregation, growing experimental evidence suggests that Fg alters vascular reactivity and impairs endothelial cell layer integrity by binding to its endothelial cell membrane receptors and activating signalling mechanisms. The purpose of this review is to discuss experimental data, which demonstrate the effects of Fg causing vascular dysfunction and to offer possible mechanisms for these effects, which could exacerbate microcirculatory complications during cardiovascular diseases accompanied by increased Fg content.

Lung ischemia-reperfusion injury: implications of oxidative stress and platelet-arteriolar wall interactions.

Abstract Pulmonary ischemia–reperfusion (IR) injury may result from trauma, atherosclerosis, pulmonary embolism, pulmonary thrombosis and surgical procedures such as cardiopulmonary bypass and lung transplantation. IR injury induces oxidative stress characterized by formation of reactive oxygen (ROS) and reactive nitrogen species (RNS). Nitric oxide (NO) overproduction via inducible nitric oxide synthase (iNOS) is an important component in the pathogenesis of IR. Reaction of NO with ROS forms RNS as secondary reactive products, which cause platelet activation and upregulation of adhesion molecules. This mechanism of injury is particularly important during pulmonary IR with increased iNOS activity in the presence of oxidative stress. Platelet–endothelial interactions may play an important role in causing pulmonary arteriolar vasoconstriction and post-ischemic alveolar hypoperfusion. This review discusses the relationship between ROS, RNS, P-selectin, and platelet–arteriolar wall interactions and proposes a hypothesis for their role in microvascular responses during pulmonary IR.

Hypothermia and Surgery: Immunologic Mechanisms for Current Practice

Objective:To examine cellular and immunologic mechanisms by which intraoperative hypothermia affects surgical patients. Summary Background Data:Avoidance of perioperative hypothermia has recently become a focus of attention as an important quality performance measure, aimed at optimizing the care of surgical patients. Anesthetized surgical patients are particularly at risk for hypothermia, which has been directly linked to the development of sequelae, such as coagulopathy, infection, morbid myocardial events, and death after surgery. However, many of the underlying immunologic mechanisms remain unclear. Methods:Venous blood samples from healthy volunteers were exposed for up to 4 hours to various temperatures following the addition of a 1 ng/mL lipopolysaccharide challenge. Innate immune function, assessed by the ability of monocytes to present antigen and coordinate cytokine release, was determined by qualitative and quantitative measurements of HLA-DR surface expression 2 hours following incubation, and proinflammatory tumor necrosis factor-α (TNF-α) and anti-inflammatory (IL-10) cytokine release in the first 4 hours. Results:Monocyte incubation at hypothermic temperatures (34°C) reduced HLA-DR surface expression, delayed TNF-α clearance, and increased IL-10 release. Conversely, hyperthermia (40°C) increased monocyte antigen presentation and resulted in rapid decay of TNF-α. However, IL-10 release was also increased. Normothermia (37°C) attenuated IL-10 release following the initial proinflammatory surge. Conclusion:Hypothermia exerts multiple effects at the cellular level, which impair innate immune function, and are associated with increased septic complications and mortality. These findings provide a physiological basis for perioperative temperature monitoring, which is a valid surgical performance measure that can be used to reduce surgical complications associated with avoidable hypothermia.

Involvement of fibrinogen specific binding in erythrocyte aggregation.

Increased fibrinogen concentration and erythrocyte aggregation are significant risk factors during various cardiovascular diseases and cerebrovascular disorders. Currently, fibrinogen‐induced erythrocyte aggregation is thought to be caused by a non‐specific binding mechanism. However, the published data on changes in erythrocyte aggregation during hypertension point to the possible existence of other mechanism(s). Therefore, we tested the hypothesis that specific binding of fibrinogen is involved in erythrocyte aggregation. It was found that Oregon Green 488‐labeled human fibrinogen specifically binds rat erythrocyte membranes with a K d of 1.3 μM. Further experiments showed that the peptide Arg‐Gly‐Asp‐Ser blocked both fibrinogen‐induced aggregation of intact erythrocytes and specific binding of fibrinogen to the erythrocyte membranes. These results suggest that in addition to non‐specific binding, a specific binding mechanism is also involved in fibrinogen‐induced erythrocyte aggregation.

Mitochondrial Mechanism of Microvascular Endothelial Cells Apoptosis in Hyperhomocysteinemia

An elevated level of homocysteine (Hcy) limits the growth and induces apoptosis. However, the mechanism of Hcy‐induced programmed cell death in endothelial cells is largely unknown. We hypothesize that Hcy induces intracellular reactive oxygen species (ROS) production that leads to the loss of transmembrane mitochondrial potential (Δψm) accompanied by the release of cytochrome‐c from mitochondria. Cytochrome‐c release contributes to caspase activation, such as caspase‐9, caspase‐6, and caspase‐3, which results in the degradation of numerous nuclear proteins including poly (ADP‐ribose) polymerase (PARP), which subsequently leads to the internucleosomal cleavage of DNA, resulting cell death. In this study, rat heart microvascular endothelial cells (MVEC) were treated with different doses of Hcy at different time intervals. Apoptosis was measured by DNA laddering and transferase‐mediated dUTP nick‐end labeling (TUNEL) assay. ROS production and MP were determined using florescent probes (2,7‐dichlorofluorescein (DCFH‐DA) and 5,5′,6,6′‐tetrachloro‐1,1′,3,3′‐tetraethyl‐benzamidazolocarbocyanin iodide (JC‐1), respectively, by confocal microscopy. Differential gene expression for apoptosis was analyzed by cDNA array. The results showed that Hcy‐mediated ROS production preceded the loss of MP, the release of cytochrome‐c, and the activation of caspase‐9 and ‐3. Moreover the Hcy treatment resulted in a decrease in Bcl2/Bax ratio, evaluated by mRNA levels. Caspase‐9 and ‐3 were activated, causing cleavage of PARP, a hallmark of apoptosis and internucleosomal DNA fragmentation. The cytotoxic effect of Hcy was blocked by using small interfering RNA (siRNA)‐mediated suppression of caspase‐9 in MVEC. Suppressing the activation of caspase‐9 inhibited the activation of caspase ‐3 and enhanced the cell viability and MP. Our data suggested that Hcy‐mediated ROS production promotes endothelial cell death in part by disturbing MP, which results in subsequent release of cytochrome‐c and activation of caspase‐9 and 3, leading to cell death. J. Cell. Biochem. 98: 1150–1162, 2006.

Balance of S1P1 and S1P2 signaling regulates peripheral microvascular permeability in rat cremaster muscle vasculature

Sphingosine-1-phosphate (S1P) regulates various molecular and cellular events in cultured endothelial cells, such as cytoskeletal restructuring, cell-extracellular matrix interactions, and intercellular junction interactions. We utilized the venular leakage model of the cremaster muscle vascular bed in Sprague-Dawley rats to investigate the role of S1P signaling in regulation of microvascular permeability. S1P signaling is mediated by the S1P family of G protein-coupled receptors (S1P(1-5) receptors). S1P(1) and S1P(2) receptors, which transduce stimulatory and inhibitory signaling, respectively, are expressed in the endothelium of the cremaster muscle vasculature. S1P administration alone via the carotid artery was unable to protect against histamine-induced venular leakage of the cremaster muscle vascular bed in Sprague-Dawley rats. However, activation of S1P(1)-mediated signaling by SEW2871 and FTY720, two agonists of S1P(1), significantly inhibited histamine-induced microvascular leakage. Treatment with VPC 23019 to antagonize S1P(1)-regulated signaling greatly potentiated histamine-induced venular leakage. After inhibition of S1P(2) signaling by JTE-013, a specific antagonist of S1P(2), S1P was able to protect microvascular permeability in vivo. Moreover, endothelial tight junctions and barrier function were regulated by S1P(1)- and S1P(2)-mediated signaling in a concerted manner in cultured endothelial cells. These data suggest that the balance between S1P(1) and S1P(2) signaling regulates the homeostasis of microvascular permeability in the peripheral circulation and, thus, may affect total peripheral vascular resistance.

Hydrogen Sulfide Mitigates Cardiac Remodeling During Myocardial Infarction via Improvement of Angiogenesis

Exogenous hydrogen sulfide (H2S) leads to down-regulation of inflammatory responses and provides myocardial protection during acute ischemia/reperfusion injury; however its role during chronic heart failure (CHF) due to myocardial infarction (MI) is yet to be unveiled. We previously reported that H2S inhibits antiangiogenic factors such, as endostatin and angiostatin, but a little is known about its effect on parstatin (a fragment of proteinase-activated receptor-1, PAR-1). We hypothesize that H2S inhibits parstatin formation and promotes VEGF activation, thus promoting angiogenesis and significantly limiting the extent of MI injury. To verify this hypothesis MI was created in 12 week-old male mice by ligation of left anterior descending artery (LAD). Sham surgery was performed except LAD ligation. After the surgery mice were treated with sodium hydrogen sulfide (30 μmol/l NaHS, a donor for H2S, in drinking water) for 4 weeks. The LV tissue was analyzed for VEGF, flk-1 and flt-1, endostatin, angiostatin and parstatin. The expression of VEGF, flk-1 and flt-1 were significantly increased in treated mice while the level of endostatin, angiostatin and parstatin were decreased compared to in untreated mice. The echocardiography in mice treated with H2S showed the improvement of heart function compared to in untreated mice. The X-ray and Doppler blood flow measurements showed enhancement of cardiac-angiogenesis in mice treated with H2S. This observed cytoprotection was associated with an inhibition of anti-angiogenic proteins and stimulation of angiogenic factors. We established that administration of H2S at the time of MI ameliorated infarct size and preserved LV function during development of MI in mice. These results suggest that H2S is cytoprotective and angioprotective during evolution of MI.

Increased erythrocyte aggregation in spontaneously hypertensive rats

Alterations of red blood cell (RBC) aggregation and plasma viscosity are major contributors to the changes in blood rheologic properties that cause an increase in peripheral vascular resistance during the development of hypertension. Although basic research and clinical study have provided considerable understanding of the pathophysiology of hypertension, the objective of this study was to determine whether an increase in RBC aggregability and plasma viscosity precede or accompany the development of high arterial blood pressure. To address this question, RBC aggregation and plasma viscosity were studied in spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto rats (WKY) at 3 and 12 weeks of age. The plasma concentrations of fibrinogen and fibronectin (FN) were also analyzed in both age groups. RBC aggregability and plasma viscosity were increased in both young and mature SHR compared to age-matched normotensive WKY rats. Mean arterial blood pressure and diastolic pressures were increased in mature hypertensive rats, whereas in young SHR only diastolic pressure was elevated significantly. The concentration of fibrinogen was higher only in the mature hypertensive rats, whereas plasma FN content was greater in both 3- and 12-week-old SHR compared to age-matched WKY. These results show the existence of increased RBC aggregability and plasma hyperviscosity not only during the established phase of hypertension, but also during the early stage of hypertension development, when mean arterial blood pressure is not yet significantly elevated in the genetically hypertensive rat model. These changes may be related to significant increase in the plasma protein FN, which occurs at the same time as the RBC aggregability and plasma viscosity changes. These results may increase attention to changes in the rheologic properties and to the mechanisms involved in these processes in the early stages of hypertension development.

Activation of GABA-A Receptor Ameliorates Homocysteine-Induced MMP-9 Activation by ERK Pathway

Hyperhomocysteinemia (HHcy) is a risk factor for neuroinflammatory and neurodegenerative diseases. Homocysteine (Hcy) induces redox stress, in part, by activating matrix metalloproteinase‐9 (MMP‐9), which degrades the matrix and leads to blood–brain barrier dysfunction. Hcy competitively binds to γ‐aminbutyric acid (GABA) receptors, which are excitatory neurotransmitter receptors. However, the role of GABA‐A receptor in Hcy‐induced cerebrovascular remodeling is not clear. We hypothesized that Hcy causes cerebrovascular remodeling by increasing redox stress and MMP‐9 activity via the extracellular signal‐regulated kinase (ERK) signaling pathway and by inhibition of GABA‐A receptors, thus behaving as an inhibitory neurotransmitter. Hcy‐induced reactive oxygen species production was detected using the fluorescent probe, 2′–7′‐dichlorodihydrofluorescein diacetate. Hcy increased nicotinamide adenine dinucleotide phosphate‐oxidase‐4 concomitantly suppressing thioredoxin. Hcy caused activation of MMP‐9, measured by gelatin zymography. The GABA‐A receptor agonist, muscimol ameliorated the Hcy‐mediated MMP‐9 activation. In parallel, Hcy caused phosphorylation of ERK and selectively decreased levels of tissue inhibitors of metalloproteinase‐4 (TIMP‐4). Treatment of the endothelial cell with muscimol restored the levels of TIMP‐4 to the levels in control group. Hcy induced expression of iNOS and decreased eNOS expression, which lead to a decreased NO bioavailability. Furthermore muscimol attenuated Hcy‐induced MMP‐9 via ERK signaling pathway. These results suggest that Hcy competes with GABA‐A receptors, inducing the oxidative stress transduction pathway and leading to ERK activation. J. Cell. Physiol. 220: 257–266, 2009.

Fibrinogen induces endothelial cell permeability

Many cardiovascular and cerebrovascular disorders are accompanied by an increased blood content of fibrinogen (Fg), a high molecular weight plasma adhesion protein. Fg is a biomarker of inflammation and its degradation products have been associated with microvascular leakage. We tested the hypothesis that at pathologically high levels, Fg increases endothelial cell (EC) permeability through extracellular signal regulated kinase (ERK) signaling and by inducing F-actin formation. In cultured ECs, Fg binding to intercellular adhesion molecule-1 and to α5β1 integrin, caused phosphorylation of ERK. Subsequently, F-actin formation increased and coincided with formation of gaps between ECs, which corresponded with increased permeability of ECs to albumin. Our data suggest that formation of F-actin and gaps may be the mechanism for increased albumin leakage through the EC monolayer. The present study indicates that elevated un-degraded Fg may be a factor causing microvascular permeability that typically accompanies cardiovascular and cerebrovascular disorders.