Role In Vascular Response Research Articles

Glycocalyx Disruption Triggers Human Monocyte Activation in Acute Heart Failure Syndromes.

Acute heart failure (AHF) syndromes manifest increased inflammation and vascular dysfunction; however, mechanisms that integrate the two in AHF remain largely unknown. The glycocalyx (GAC) is a sugar-based shell that envelops all mammalian cells. Much GAC research has focused on its role in vascular responses, with comparatively little known about how the GAC regulates immune cell function. In this study, we sought to determine if GAC degradation products are elevated in AHF patients, how these degradation products relate to circulating inflammatory mediators, and whether the monocyte GAC (mGAC) itself modulates monocyte activation. Inflammatory markers and GAC degradation products were profiled using ELISAs. Flow cytometry was used to assess the mGAC and RNA-seq was employed to understand the role of the mGAC in regulating inflammatory activation programs. In a cohort of hospitalized AHF patients (n = 17), we found that (1) the GAC degradation product heparan sulfate (HS) was elevated compared with age-matched controls (4396 and 2903ng/mL; p = 0.01) and that (2) HS and soluble CD14 (a marker of monocyte activation) levels were closely related (Pearson's r = 0.65; p = 0.002). Mechanistically, Toll-like receptor (TLR) activation of human monocytes results in GAC remodeling and a decrease in the mGAC (71% compared with no treatment; p = 0.0007). Additionally, we found that ex vivo enzymatic removal of HS and disruption of the mGAC triggers human monocyte activation and amplifies monocyte inflammatory responses. Specifically, using RNA-seq, we found that enzymatic degradation of the mGAC increases transcription of inflammatory (IL6, CCL3) and vascular (tissue factor/F3) mediators. These studies indicate that the mGAC is dynamically remodeled during monocyte activation and that mGAC remodeling itself may contribute to the heightened inflammation associated with AHF.

Perspectives for angiotensin profiling with liquid chromatography/mass spectrometry to evaluate ACE/ACE2 balance in endothelial dysfunction and vascular pathologies.

Vascular injury, characterized by endothelial dysfunction, inflammation, structural remodeling, thrombosis and calcification leads to cardiovascular diseases. Angiotensin (Ang) II (1-8) - synthesized mainly by angiotensin converting enzyme (ACE) is the best characterized mediator of the renin-angiotensin system (RAS). This peptide initially identified by its vasoactive properties was found to play a major role in vascular response to insult. However, recent discovery of angiotensin converting enzyme 2 (ACE2) that produces vasoprotective Ang-(1-7) peptide highlighted complexity of the system and suggested that balance between ACE/Ang II and ACE2/Ang-(1-7) is fundamental in maintaining vascular homeostasis and its disorders are associated with cardiovascular pathology. There is therefore a need to develop methods for comprehensive analysis of biologically active Ang peptides and their metabolites of ACE/Ang II and ACE2/Ang-(1-7) axes. Liquid chromatography/mass spectrometry (LC/MS) is an analytical technique that offers potential for specific, simultaneous analysis of Ang peptides. With sensitivity added by application of preconcentration nanochromatography reaching picomolar concentrations, practically all Ang peptides identified so far could be quantified in biological samples. Ang profiling is important not only for understanding their physiological or pathological role but could also serve as an early diagnostic biomarker of endothelial dysfunction and cardiovascular pathology. It could also be used for monitoring the efficacy of the RAS-targeted therapies. Although, the methodology requires further improvements to adopt it for routine application, Ang peptide profiling with targeted LC/MS analysis might assess functional balance between ACE/Ang II and ACE2/Ang-(1-7) axes, facilitate our understanding of the cardiovascular pathology and enhance biomarker portfolio in cardiovascular diseases.

Polymorphonuclear neutrophils and instability of the atherosclerotic plaque: a causative role?

The aim of this review is to examine the role of polymorphonuclear neutrophils (PMNs) in the evolution of atherosclerosis. While the role of PMNs in the evolution of atherosclerosic process has failed until recently to attract much attention, a body of research carried out over the last decade has disclosed the unexpectedly complex behavior of these cells, unraveling an unexpected key role for PMNs in the onset and progression of atheroma. A PubMed database search was performed for studies providing evidences on the role of PMNs in the development and progression of atherosclerotic lesion. Activated PMNs were shown to produce and release reactive oxygen species, inflammatory leukotrienes and proteolytic lysosomal enzymes, directly inducing vascular damage. Activated PMNs also secrete myeloperoxidase, involved in lipoprotein oxidation. PMNs have a finite lifespan and typically die through apoptosis, which thus represents a counter-regulatory mechanism limiting the toxic potential of these short-lived, terminally differentiated cells. Dysregulation of this process probably contributes to the pathogenesis and progression of several inflammatory diseases. Moreover, high circulating levels of PMN-platelet aggregates have been reported in patients with clinical atherosclerosis, and recent studies suggest that these aggregates may play a role in vascular response to injury. It has been suggested that this heterotypic interaction between platelets and leukocytes might represent a link between hemostasis/thrombosis and the inflammatory response.

The dorsomedial hypothalamus (DMH) plays a strain-dependent role in vascular responses to cocaine but not cold stress in conscious rats

s / Autonomic Neuroscience: Basic and Clinical 163 (2011) 1–133 56 P.039 The dorsomedial hypothalamus (DMH) plays a strain-dependent role in vascular responses to cocaine but not cold stress in conscious rats M.M. Knuepfer, M. Busauskas (St. Louis University School of Medicine Pharmacol. & Physiol. Science, USA) We demonstrated that cocaine or cold water stress (CWS) elicits systemic vasoconstriction in Brown-Norway (BN) rats but an increase in cardiac output in Dahl salt sensitive (DSS) rats. Since the DMH plays an important role in stress responsiveness, we sought to determine the role of the DMH to these disparate stressors on regional vascular hemodynamics. We hypothesized that inhibition of the DMH would attenuate greater hindquarters vasoconstriction to either stress in BN compared to DSS rats. BN and DSS rats were instrumented for determination of arterial pressure, heart rate, andwith pulsed Doppler flowprobes on the superiormesenteric and abdominal aorta. Bilateral guide cannulaswere implanted above the DMH. After recovery, we observed that cocaine (5 mg/kg, iv) elicited greater hindquarters andmesenteric vasoconstriction and greater bradycardia in BN rats compared toDSS rats. In contrast, CWS (1 cm deep for 1 min) elicited tachycardia in all rats and greater increases in mesenteric resistance in BN rats but hindquarters vasodilation that was greater in BN compared to DSS rats. Therefore, visceral and skeletalmuscle vascular responses differed between strains and stressors. We examined the dependence of these responses on the DMH with bilateral microinjections of muscimol (80 pmol in 100 nl) in theDMH.Muscimol attenuated thegreaterhindquarters andmesenteric vasoconstriction and tachycardia in BN rats without affecting responses in DSS rats in response to cocaine. Responses to CWSwere unaffected by muscimol except that tachycardia was selectively reduced in BN rats. Kainate (10 pmol) in the DMH elicited a similar response pattern as CWS. These studies demonstrate that the DMH plays a strainand stimulus-dependent role in mediating hemodynamic responses to stress. Our data suggest that there are significant variations in central neural pathways mediating stress that vary in different rat strains.

A2B adenosine receptor dampens hypoxia-induced vascular leak

Extracellular adenosine has been implicated in adaptation to hypoxia and previous studies demonstrated a central role in vascular responses. Here, we examined the contribution of individual adenosine receptors (ARs: A1AR/A2AAR/A2BAR/A3AR) to vascular leak induced by hypoxia. Initial profiling studies revealed that siRNA-mediated repression of the A2BAR selectively increased endothelial leak in response to hypoxia in vitro. In parallel, vascular permeability was significantly increased in vascular organs of A2BAR(-/-)-mice subjected to ambient hypoxia (8% oxygen, 4 hours; eg, lung: 2.1 +/- 0.12-fold increase). By contrast, hypoxia-induced vascular leak was not accentuated in A1AR(-/-)-, A2AAR(-/-)-, or A3AR(-/-)-deficient mice, suggesting a degree of specificity for the A2BAR. Further studies in wild type mice revealed that the selective A2BAR antagonist PSB1115 resulted in profound increases in hypoxia-associated vascular leakage while A2BAR agonist (BAY60-6583 [2-[6-amino-3,5-dicyano-4-[4-(cyclopropylmethoxy)-. phenyl]pyridin-2-ylsulfanyl]acetamide]) treatment was associated with almost complete reversal of hypoxia-induced vascular leakage (eg, lung: 2.0 +/- 0.21-fold reduction). Studies in bone marrow chimeric A2BAR mice suggested a predominant role of vascular A2BARs in this response, while hypoxia-associated increases in tissue neutrophils were, at least in part, mediated by A2BAR expressing hematopoietic cells. Taken together, these studies provide pharmacologic and genetic evidence for vascular A2BAR signaling as central control point of hypoxia-associated vascular leak.

Comparative effects of pitavastatin and probucol on oxidative stress, Cu/Zn superoxide dismutase, PPAR-γ, and aortic stiffness in hypercholesterolemia

Reactive oxygen species-scavenging enzyme Cu/Zn superoxide dismutase (SOD) regulated by peroxisome proliferator-activated receptors (PPARs) plays an important role in vascular responsiveness. However, it remains unknown whether statin restores vascular dysfunction through the activation of reactive oxygen species-scavenging enzymes in vivo. We hypothesized that pitavastatin restores vascular function by modulating oxidative stress through the activation of Cu/ZnSOD and PPAR-gamma in hypercholesterolemia. New Zealand White male rabbits were fed either normal chow or a 1% cholesterol (CHO) diet for 14 wk. After the first 7 wk, the CHO-fed rabbits were further divided into three groups: those fed with CHO feed only (HC), those additionally given pitavastatin, and those additionally given an antioxidant, probucol. The extent of atherosclerosis was assessed by examining aortic stiffness. When compared with the HC group, both the pitavastatin and probucol groups showed improved aortic stiffness by reducing aortic levels of reactive oxidative stress, nitrotyrosine, and collagen, without affecting serum cholesterol or blood pressure levels. Pitavastatin restored both Cu/ZnSOD activity (P < 0.005) and PPAR-gamma expression and activity (P < 0.01) and inhibited NAD(P)H oxidase activity (P < 0.0001) in the aorta, whereas probucol inhibited NAD(P)H oxidase activity more than did pitavastatin (P < 0.0005) without affecting Cu/ZnSOD activity or PPAR-gamma expression and activity. Importantly, Cu/ZnSOD activity was positively correlated with the PPAR-gamma activity in the aorta (P < 0.005), both of which were negatively correlated with aortic stiffness (P < 0.05). Vascular Cu/ZnSOD and PPAR-gamma may play a crucial role in the antiatherogenic effects of pitavastatin in hypercholesterolemia in vivo.

Thrombin and PAR-1 stimulate differentiation of bone marrow-derived endothelial progenitor cells.

Endothelial progenitor cells (EPCs) from the bone marrow play an important role in vascular response to injury and ischemia. The mediators involved in the mobilization, recruitment, proliferation and differentiation of EPCs are not fully understood. In this study, the role of coagulation factor thrombin and protease-activated receptor-1 (PAR-1) on bone marrow-derived cell proliferation and differentiation was investigated. Bone marrow cells (BMCs) were isolated from C57/BL6 mice and plated on fibronectin-coated flasks. Cell characteristics, proliferation and the expression of endothelial cell markers were determined using immunohistochemistry, thymidine uptake and fluorescence activated-cell sorting (FACS), respectively. The results show that thrombin stimulated enrichment of bone marrow cells with endothelial morphology, exhibiting acetylated-low-density lipoprotein (LDL) uptake and isolectin staining. Thrombin or PAR-1-activating peptide produced a 2- to 3-fold increase in the total number of cells as well as an increase in vascular endothelial (VE)-cadherin-positive cells. Thrombin treatment of VE-cadherin-negative cells prepared after cell sorting resulted in the generation of 3- to 4-fold higher VE-cadherin-positive cells than the untreated cultures. Increase in VE-cadherin-positive cells was inhibited by hirudin and efegatran. These results provide first evidence for a novel activity of thrombin and PAR-1 on bone marrow progenitor cell proliferation and EPC differentiation, and suggest their potential role in vascular regeneration and recanalization of thrombus.

Transcriptional regulation of the CRLR gene in human microvascular endothelial cells by hypoxia.

Adrenomedullin is a 52 amino acid peptide that shows a remarkable range of effects on the vasculature that include inter alia, vasodilatation, regulation of permeability, inhibition of endothelial cell apoptosis, and promotion of angiogenesis. Recently the G-protein coupled receptor (GPCR) calcitonin receptor-like receptor (CRLR), and receptor activity modifying proteins (RAMPs) have become recognized as integral components of the adrenomedullin signaling system. However, mechanisms of regulation of CRLR expression are still largely unknown. This is in part due to lack of information on the gene promoter. In this study we have determined the transcriptional start of human CRLR cDNA by 5'-RACE and cloned the proximal 5'-flanking region of the gene by PCR. The 2318 bp genomic fragment contains the basal promoter of human CRLR, including potential TATA-boxes and several GC boxes. Regulatory elements binding known transcription factors, such as Sp-1, Pit-1, glucocorticoid receptor, and hypoxia-inducible factor-1 alpha (HIF-1alpha) were also identified. When cloned into reporter gene vectors, the genomic fragment showed significant promoter activity, indicating that the 5'-flanking region isolated by PCR contains the gene promoter of human CRLR. Of significance is that the cloned promoter fragments were activated by hypoxia when transfected in primary microvascular endothelial cells. Site-directed mutagenesis of the consensus hypoxia-response element (HRE) in the 5'-flanking region abolished such a response. We also demonstrated by semi-quantitative RT-PCR that transcription of the gene is activated by hypoxia in microvascular endothelial cells. In contrast, expression of RAMPs 1, 2, and 3 was unaffected by low oxygen tension. We conclude that simultaneous transcriptional up-regulation of CRLR and its ligand adrenomedullin in endothelial cells could lead to a potent survival loop and therefore might play a significant role in vascular responses to hypoxia and ischemia.

Response to hypoxia involves transforming growth factor-β2 and Smad proteins in human endothelial cells

AbstractOxygen deprivation (hypoxia) is a consistent component of ischemia that induces an inflammatory and prothrombotic response in the endothelium. In this report, it is demonstrated that exposure of endothelial cells to hypoxia (1% O2) increases messenger RNA and protein levels of transforming growth factor-β2 (TGF-β2), a cytokine with potent regulatory effects on vascular inflammatory responses. Messenger RNA levels of the TGF-β2 type II membrane receptor, which is a serine threonine kinase, also increased. The stimulatory effect of hypoxia was found to occur at the level of transcription of the TGF-β2 gene and involves Smad proteins, a class of intracellular signaling proteins that mediates the downstream effects of TGF-β receptors. Transient transfection studies showed that the region spanning −77 and −40 base pairs within the TGF-β2 promoter (harboring a Smad-binding “CAGA box”) is activated in hypoxic cells compared with nonhypoxic controls (P < .01). Hypoxia also stimulated transcription from another promoter, 3TP-Lux, a reporter construct responsive to Smads and TGF-β. In addition, specific binding to a Smad-binding oligonucleotide was observed with nuclear extracts from hypoxic endothelial cells but not from nonhypoxic cells. It is concluded that Smad proteins, which can regulate endothelial responses to mechanical and inflammatory stress, also may play an important role in vascular responses to hypoxia and ischemia.

New Mechanisms for Inhaled NO

New Mechanisms for Inhaled NO

Sp1-mediated downregulation of P2X4 receptor gene transcription in endothelial cells exposed to shear stress.

Endothelial purinoceptors play an important role in vascular responses to extracellular adenine nucleotides and hemodynamic forces. Here we report that P2X4 purinoceptor expression in human umbilical vein endothelial cells is transcriptionally downregulated by fluid shear stress. When human umbilical vein endothelial cells were subjected to a laminar shear stress of 15 dyn/cm(2), P2X4 mRNA levels began to decrease within 1 h and further decreased with time, reaching 60% at 24 h. Functional analysis of the 1.9-kb P2X4 5'-promoter indicated that a 131-bp segment (-112 to +19 bp relative to the transcription start site) containing a consensus binding site for the Sp1 transcription factor was critical for the shear stress responsiveness. Mutations of the Sp1 site decreased the basal level of transcription and abolished the response of the P2X4 promoter to shear stress. Electrophoretic mobility shift assays showed a marked decrease in binding of Sp1 to the Sp1 consensus element in shear-stressed cells, suggesting that Sp1 mediates the shear stress-induced downregulation of P2X4 gene transcription.

Hypoxia increases cytosolic free calcium in porcine pulmonary arterial endothelial cells.

Alteration of cytosolic free calcium ([Ca2+]i) in hypoxia was studied with fluorescent probe, Fura-2 in cultured porcine pulmonary arterial endothelial cells. It was found that hypoxia caused by bubbling the cell suspension with 100% N2 increased [Ca2+]i in endothelial cells by 90 +/- 20% (n = 8, P < 0.05), but didn't affect that in a Ca(2+)-free buffer, La3+ (2 x 10(-5) mol/L) inhibited the hypoxia-induced increment in [Ca2+]i, whereas verapamil (10(-8) mol/L) didn't. These findings suggest that hypoxia could induce Ca2+ influx in pulmonary arterial endothelial cells, which might play an important role in vascular response to hypoxia.

Uptake of norepinephrine in an isolated artery from normotensive humans.

Previous studies have suggested that catecholamine uptake may play a role in vascular responsiveness in hypertension. The current study was undertaken to characterize amine uptake and effects of its inhibition in an isolated human artery from normotensive subjects and to provide a basis for future study in hypertensive individuals. Accumulation of tritiated norepinephrine into the artery of normotensive subjects was time-dependent and 16.9-fold more than the incubation media concentration (1 microM) of amine after 60 minutes. There was a lesser accumulation of tritiated normetanephrine (3.1-fold), and it was not increased over time. Increasing the concentration of norepinephrine to 30 microM did not significantly change the proportional accumulation. Inhibition of neuronal uptake with cocaine (10 microM) reduced the average accumulation of both concentrations of tritiated norepinephrine to 3.9-fold (p less than 0.001). Inhibition of extraneuronal uptake with corticosterone alone (10 microM) had no significant effect on average accumulation of norepinephrine, and where combined with cocaine, there was no further effect of corticosterone. Neither cocaine nor corticosterone had any effects on accumulation of normetanephrine. In spite of elimination of approximately 75% of the uptake of norepinephrine, cocaine had very little potentiating effect on mechanical responses to exogenous norepinephrine and neurally released transmitter. Thus, norepinephrine uptake in human cystic artery is characteristic of neuronal uptake, but cocaine treatment has only a very modest potentiating effect on responsiveness to endogenous norepinephrine and no significant effect on responsiveness to exogenous amine.