Vascular Cell Activation Research Articles

Suppression of Gut Bacterial Translocation Ameliorates Vascular Calcification through Inhibiting Toll-Like Receptor 9-Mediated BMP-2 Expression.

Aims Vascular calcification (VC) is a primary risk factor for cardiovascular mortality in chronic renal failure (CRF) patients; thus, effective therapeutic targets are urgently needed to be explored. Here, we identified the role of intestinal bacterial translocation in CRF-related VC. Methods and Results Antibiotic supplementation by oral gavage significantly suppressed intestinal bacterial translocation, CRF-related VC, and aortic osteogenic gene and Toll-like receptor (TLR) gene expression in CRF rats. Furthermore, TLR4 and TLR9 activation in vascular smooth muscle cells (VSMCs) aggravated inorganic phosphate- (Pi-) induced calcification. TLR9 inhibition, but not TLR4 inhibition, by both a pharmacological inhibitor and genetic methods could significantly reduce CRF rats' serum or CRF-induced VC. Interestingly, bone morphogenic protein-2 (BMP-2) levels were increased in the aorta and sera from CRF rats. Increased BMP-2 levels were also observed in VSMCs treated with TLR9 agonist, which was blocked by NF-κB inhibition. Both siRNA knockdown of BMP-2 and NF-κB inhibitor significantly blocked TLR9 agonist-induced VSMC calcification. Conclusions Gut bacterial translocation inhibited by oral antibiotic significantly reduces CRF-related VC through inhibition of TLR9/NF-κB/BMP-2 signaling.

Protein kinase B/AKT mediates insulin-like growth factor 1-induced phosphorylation and nuclear export of histone deacetylase 5 via NADPH oxidase 4 activation in vascular smooth muscle cells.

Insulin-like growth factor 1 (IGF-1) mediates the generation of reactive oxygen species (ROS) and the activation of growth promoting signaling pathways. Histone deacetylases (HDACs) regulate gene transcription by deacetylating lysine residues in histone and nonhistone proteinsand a heightened HDAC activation, notably of HDAC5, is associated with vascular disorders, such as atherosclerosis. Although the contribution of IGF-1 in these pathologies is well documented, its role in HDAC phosphorylation and activation remains unexplored. Here, we examined the effect of IGF-1 on HDAC5 phosphorylation in vascular smooth muscle cells (VSMCs) and identified the signaling pathways involved in controlling HDAC5 phosphorylation and nuclear export. Treatment of A10 VSMCs with IGF-1 enhanced HDAC5 phosphorylation. Blockade of the IGF-1 receptor tyrosine kinase (TK) activity with the specific pharmacological inhibitor, AG1024, significantly inhibited IGF-1-induced HDAC5 phosphorylation, whereas the epidermal growth factor receptor (EGFR) TK antagonist, AG1478, had no effect. Inhibition of the mitogen-activated protein kinase pathway with U0126, SP600125, or SB203580, did not affect HDAC5 phosphorylation, whereas two inhibitors of the phosphoinositide 3-kinase (PI3K)/AKT pathways, wortmannin and SC66, almost completely attenuated IGF-1-induced responses as confirmed by immunoblotting of phospho-HDAC5 and by small interfering RNA (siRNA)-induced AKT silencing. Moreover, the NAD(P)H oxidase (Nox) inhibitor, diphenyleneiodonium (DPI), and Nox4 siRNA, attenuated IGF-1-induced phosphorylation of HDAC5 and AKT. The HDAC5 phosphorylation resulted in its nuclear export, which was reversed by SC66 and DPI. Our results indicate that IGF-1-induced phosphorylation and nuclear export of HDAC5 involve Nox4-dependent ROS generation and PI3K/AKT signaling pathways.

IL-1β/MMP9 activation in primary human vascular smooth muscle-like cells: Exploring the role of TNFα and P2X7

BackgroundVascular smooth muscle cells exhibit phenotypic plasticity in response to microenvironmental stimuli and contribute to vascular remodelling through mechanisms only partially understood.In atherosclerosis, P2X-purinoceptor7 (P2X7) has been related to interleukin-1β (IL-1β) and metalloproteinase 9 (MMP9). The hypoxia-inducible factor-1alpha (HIF1α) was associated to remodelling. Here the activation of IL-1β and MMP9 was studied in relationship to P2X7 and HIF1α in cells exploited from human carotid plaque and internal mammary artery. Methods and resultsMigrating cells expressed HIF1α-regulated canopy FGF-signalling regulator 2 and CD117, and led to primary cells with SMC-like phenotype (VSMC), P2X7+.We investigated in VSMC the effects of hypoxia, of treatment with tumour necrosis factor-α (TNFα) and/or with P2X7 antagonist, A740003. Quantitative RT-PCR showed that hypoxia unaffected IL-1β and down-regulated MMP9 mRNAs, without activating HIF1α. TNFα increased IL-1β mRNA via NLR Family Pyrin Domain-Containing 3, with production of proIL-1β but no rise of mature IL-1β. Zymography demonstrated that A740003 triggered MMP9 secretion from VSMC. Combination of A740003 with TNFα abrogated this effect. Combination was ineffective on IL-1β activation elicited by TNFα, but down-regulated HIF1α mRNA. A740003 induced the intracellular P2X7 aggregation and differently perturbed lysosome and mitochondria network compared to TNFα. ConclusionsCells migration from human arteries leads to partially differentiated VSMC analogous to neointimal cells within atherosclerotic lesions. Down-regulated HIF1α in stimulated VSMC translates in resilience in atherosclerotic lesions. P2X7-independent partial activation of IL-1β elicited by TNFα underlines complexity of the cytokine secretion. Data also supported P2X7 as modulator of MMP9 secretion, important for atherosclerosis progression.

NOXA1-dependent NADPH oxidase regulates redox signaling and phenotype of vascular smooth muscle cell during atherogenesis

Increased reactive oxygen species (ROS) production and inflammation are key factors in the pathogenesis of atherosclerosis. We previously reported that NOX activator 1 (NOXA1) is the critical functional homolog of p67phox for NADPH oxidase activation in mouse vascular smooth muscle cells (VSMC). Here we investigated the effects of systemic and SMC-specific deletion of Noxa1 on VSMC phenotype during atherogenesis in mice.Neointimal hyperplasia following endovascular injury was lower in Noxa1-deficient mice versus the wild-type following endovascular injury. Noxa1 deletion in Apoe-/- or Ldlr-/- mice fed a Western diet showed 50% reduction in vascular ROS and 30% reduction in aortic atherosclerotic lesion area and aortic sinus lesion volume (P < 0.01). SMC-specific deletion of Noxa1 in Apoe-/- mice (Noxa1SMC-/-/Apoe-/-) similarly decreased vascular ROS levels and atherosclerotic lesion size. TNFα-induced ROS generation, proliferation and migration were significantly attenuated in Noxa1-deficient versus wild-type VSMC. Immunofluorescence analysis of atherosclerotic lesions showed a significant decrease in cells positive for CD68 and myosin11 (22% versus 9%) and Mac3 and α-actin (17% versus 5%) in the Noxa1SMC-/-/Apoe-/- versus Apoe-/- mice. The expression of transcription factor KLF4, a modulator of VSMC phenotype, and its downstream targets – VCAM1, CCL2, and MMP2 – were significantly reduced in the lesions of Noxa1SMC-/-/Apoe-/- versus Apoe-/- mice as well as in oxidized phospholipids treated Noxa1SMC-/- versus wild-type VSMC.Our data support an important role for NOXA1-dependent NADPH oxidase activity in VSMC plasticity during restenosis and atherosclerosis, augmenting VSMC proliferation and migration and KLF4-mediated transition to macrophage-like cells, plaque inflammation, and expansion.

Subcellular Localization and Activity of the Mitogen-Activated Protein Kinase Kinase 7 (MKK7) γ Isoform are Regulated through Binding to the Phosphatase Calcineurin.

Calcineurin (CaN) phosphatase signaling is regulated by targeting CaN to substrates, inhibitors, and scaffold proteins containing docking motifs with the consensus sequence of PxIxIT. Here, we identify the docking of CaN to the γ isoform of MKK7, a component of the c-Jun N-terminal kinase (JNK) pathway. Because of alternative splicing of a single exon within the N-terminal domain, MKK7γ encodes a unique PxIxIT motif (PIIVIT) that is not present in MKK7α or β We found that MKK7γ bound directly to CaN through this PIIVIT motif in vitro, immunoprecipitated with CaN from cell extracts, and exhibited fluorescence resonance energy transfer (FRET) with CaN in the cytoplasm but not in the nucleus of living cells. In contrast, MKK7α and β exhibited no direct binding or FRET with CaN and were localized more in the nucleus than the cytoplasm. Furthermore, the inhibition of CaN phosphatase activity increased the basal phosphorylation of MKK7γ but not MKK7β Deletion of the MKK7γ PIIVIT motif eliminated FRET with CaN and promoted MKK7γ redistribution to the nucleus; however, the inhibition of CaN activity did not alter MKK7γ localization, indicating that MKK7γ cytoplasmic retention by CaN is phosphatase activity independent. Finally, the inhibition of CaN phosphatase activity in vascular smooth muscle cells, which express MKK7γ mRNA, enhances JNK activation. Overall, we conclude that the MKK7γ-specific PxIxIT motif promotes high-affinity CaN binding that could promote novel cross talk between CaN and JNK signaling by limiting MKK7γ phosphorylation and restricting its localization to the cytoplasm.

Pivotal Pathogenic and Biomarker Role of Chlamydia Pneumoniae in Neurovascular Diseases.

Chlamydia Pneumoniae (C. Pn) is an obligatory intracellular bacterium that is associated with respiratory tract infections like pneumonia, pharyngitis and bronchitis. It has also been implicated in cerebrovascular (stroke) as well as cardiovascular diseases. The most possible pathway via which C. Pn elicits its pathogenesis could be via activation of Human Vascular Smooth Muscle Cells (VSMCs) proliferation resulting in the stimulation of Toll-Like Receptor-4 (TLR-4) and/or phospho-44/42(p44/p42) Mitogen-Activated Protein Kinases (MAPK). It is also established that tyrosine phosphorylation of IQ domain GTPase-activating protein 1 (IQGAP1) also contributed to C. Pn infection-triggered Vascular Endothelial Cell (VEC) movements via the SRC tyrosine kinase inhibitor PP2 (4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine) resulting in angiogenesis. It is also proven that restricted inflammatory cell infiltrates as well as apoptosis have been linked to C. Pn or C. Pn-specific proteins in atherosclerotic plaques of patients with stroke. It is further an evidence that C. Pn enters the cerebral vasculature during the initial infection and worsen atherosclerosis either directly or indirectly. Chronic, persistent C. Pn infection is also capable of triggering the secretion of Chlamydial Heat Shock Protein 60 (cHSP60) in the vessel wall resulting in augmentation of inflammation. C. Pn also aids in the activation of explicit cell-intermediated immunity within plaques. Macrophages in the carotid plaques co-exist with CD4+ lymphocytes which are capable of triggering the release of pro-inflammatory cytokines resulting in the augmentation of atherogenic development during C. Pn infection. C. Pn actively participated in the modification of both histones H3 and H4 during chromatin analysis via the interleukin 8(IL-8) gene facilitator as well as conscription of nuclear factor kappa-B(NF-κB) or NF- κB/p65 complex and polymerase II (Pol II). This review, therefore, focuses on the crucial involvement of C. Pn in the pathogenesis of cerebrovascular events.

Examining the Role of and Treatment Directed at IL-1β in Atherosclerosis.

The purpose of this review was to examine the role of IL-1β in the inflammatory process central to the development of atherosclerosis and to discuss current clinical evidence for treatments targeting IL-1β in coronary artery disease. IL-1β has been shown to modulate atherosclerotic plaque progression by upregulating the synthesis of adhesion molecules on endothelial cells, as well increasing activation and proliferation of vascular smooth muscle cells. Animal studies have further suggested that alterations in the balance between agonists and antagonists of IL-1β are important in promoting atherosclerosis. In humans, preliminary assessment of therapy targeting IL-1β noted early reductions in serum inflammatory biomarkers among those with systemic inflammatory or coronary artery disease. The CANTOS trial, a large randomized double-blind study found that canakinumab, a monoclonal antibody targeting IL-1β, reduced ischemic events in patients being treated for secondary prevention. Cellular, animal, and now clinical studies have suggested a role for therapies aimed at IL-1β for treatment of CAD. However, given potential side effects and costs of these medications, further study is required to determine which patients may be most suited for treatment above current standard of care.

Lysosomal membrane permeabilization causes secretion of IL-1\u03b2 in human vascular smooth muscle cells

ObjectiveIL-1β secretion by the inflammasome is strictly controlled and requires two sequential signals: a priming signal and an activating signal. Lysosomal membrane permeabilization (LMP) plays a critical role in the regulation of NLRP3 inflammasome, and generally acts as an activating signal. However, the role of LMP controlling NLRP3 inflammasome activation in human vascular smooth muscle cells (hVSMCs) is not well defined.MethodsLMP was induced in hVSMCs by Leu-Leu-O-methyl ester. Cathepsin B was inhibited by CA-074 Me. Cytokine release, mRNA, and protein were quantified by enzyme-linked immunosorbent assay, quantitative PCR, and Western blot, respectively. NF-κB activity was analyzed by immunostaining of the NF-κB p65 nuclear translocation and using the dual-luciferase reporter assay system.ResultsLMP had both priming and activating roles, causing an upregulation of proIL-1β and NLRP3 and the secretion of mature IL-1β from unprimed hVSMCs. LMP activated the canonical NF-κB pathway. The priming effect of LMP was inhibited by CA-074 Me, indicating an upstream role of cathepsin B.ConclusionsThese data support a novel role of LMP as a single stimulus for the secretion of IL-1β from hVSMCs, implying the possibility that hVSMCs are an important initiator of the sterile inflammatory response caused by lysosomal disintegration.

HIF-1 activation in vascular smooth muscle cells triggers osteochondrogenic reprogramming and contributes to vascular calcification

HIF-1 activation in vascular smooth muscle cells triggers osteochondrogenic reprogramming and contributes to vascular calcification

Nrf2 Activation Provides Atheroprotection in Diabetic Mice Through Concerted Upregulation of Antioxidant, Anti-inflammatory, and Autophagy Mechanisms.

Interactive relationships between metabolism, inflammation, oxidative stress, and autophagy in the vascular system play a key role in the pathogenesis of diabetic cardiovascular disease. Nuclear factor (erythroid-derived 2)-like 2 (Nrf2) is a stress-sensitive guarantor of cellular homeostasis, which cytoprotective contributions extend beyond the antioxidant defense. We investigated the beneficial effects and underlying mechanisms of the Nrf2 inducer tert-butyl hydroquinone (tBHQ) on diabetes-driven atherosclerosis. In the experimental model of streptozotocin-induced diabetes in apolipoprotein E-deficient mice, treatment with tBHQ increased Nrf2 activity in macrophages and vascular smooth muscle cells within atherosclerotic lesions. Moreover, tBHQ significantly decreased the size, extension and lipid content of atheroma plaques, and attenuated inflammation by reducing lesional macrophages (total number and M1/M2 phenotype balance), foam cell size and chemokine expression. Atheroprotection was accompanied by both systemic and local antioxidant effects, characterized by lower levels of superoxide anion and oxidative DNA marker 8-hydroxy-2′-deoxyguanosine, reduced expression of NADPH oxidase subunits, and increased antioxidant capacity. Interestingly, tBHQ treatment upregulated the gene and protein expression of autophagy-related molecules and also enhanced autophagic flux in diabetic mouse aorta. In vitro, Nrf2 activation by tBHQ suppressed cytokine-induced expression of pro-inflammatory and oxidative stress genes, altered macrophage phenotypes, and promoted autophagic activity. Our results reinforce pharmacological Nrf2 activation as a promising atheroprotective approach in diabetes, according to the plethora of cytoprotective mechanisms involved in the resolution of inflammation and oxidative stress, and restoring autophagy.

Mechanisms of PKA-Dependent Potentiation of Kv7.5 Channel Activity in Human Airway Smooth Muscle Cells.

β-adrenergic receptor (βAR) activation promotes relaxation of both vascular and airway smooth muscle cells (VSMCs and ASMCs, respectively), though the signaling mechanisms have not been fully elucidated. We previously found that the activity of Kv7.5 voltage-activated potassium channels in VSMCs is robustly enhanced by activation of βARs via a mechanism involving protein kinase A (PKA)-dependent phosphorylation. We also found that enhancement of Kv7 channel activity in ASMCs promotes airway relaxation. Here we provide evidence that Kv7.5 channels are natively expressed in primary cultures of human ASMCs and that they conduct currents which are robustly enhanced in response to activation of the βAR/cyclic adenosine monophosphate (cAMP)/PKA pathway. MIT Scansite software analysis of putative PKA phosphorylation sites on Kv7.5 identified 8 candidate serine or threonine residues. Each residue was individually mutated to an alanine to prevent its phosphorylation and then tested for responses to βAR activation or to stimuli that elevate cAMP levels. Only the mutation of serine 53 (S53A), located on the amino terminus of Kv7.5, significantly reduced the increase in Kv7.5 current in response to these stimuli. A phospho-mimic mutation (S53D) exhibited characteristics of βAR-activated Kv7.5. Serine-to-alanine mutations of 6 putative PKA phosphorylation sites on the Kv7.5 C-terminus, individually or in combination, did not significantly reduce the enhancement of the currents in response to forskolin treatment (to elevate cAMP levels). We conclude that phosphorylation of S53 on the amino terminus of Kv7.5 is essential for PKA-dependent enhancement of channel activity in response to βAR activation in vascular and airway smooth muscle cells.

Mitochondrial uncoupler BAM15 inhibits artery constriction and potently activates AMPK in vascular smooth muscle cells

Our previous studies found that mitochondrial uncouplers CCCP and niclosamide inhibited artery constriction and the mechanism involved AMPK activation in vascular smooth muscle cells. BAM15 is a novel type of mitochondrial uncoupler. The aim of the present study is to identify the vasoactivity of BAM15 and characterize the BAM15-induced AMPK activation in vascular smooth muscle cells (A10 cells). BAM15 relaxed phenylephrine (PE)-induced constricted rat mesenteric arteries with intact and denuded endothelium. Pretreatment with BAM15 inhibited PE-induced constriction of rat mesenteric arteries with intact and denuded endothelium. BAM15, CCCP, and niclosamide had the comparable IC50 value of vasorelaxation in PE-induced constriction of rat mesenteric arteries. BAM15 was less cytotoxic in A10 cells compared with CCCP and niclosamide. BAM15 depolarized mitochondrial membrane potential, induced mitochondrial fission, increased mitochondrial ROS production, and increased mitochondrial oxygen consumption rate in A10 cells. BAM15 potently activated AMPK in A10 cells and the efficacy of BAM15 was stronger than that of CCCP, niclosamide, and AMPK positive activators metformin and AICAR. In conclusion, BAM15 activates AMPK in vascular smooth muscle cells with higher potency than that of CCCP, niclosamide and the known AMPK activators metformin and AICAR. The present work indicates that BAM15 is a potent AMPK activator.

CRISPR/Cas9-mediated gene knockout reveals a guardian role of NF-\u03baB/RelA in maintaining the homeostasis of human vascular cells

Vascular cell functionality is critical to blood vessel homeostasis. Constitutive NF-κB activation in vascular cells results in chronic vascular inflammation, leading to various cardiovascular diseases. However, how NF-κB regulates human blood vessel homeostasis remains largely elusive. Here, using CRISPR/Cas9-mediated gene editing, we generated RelA knockout human embryonic stem cells (hESCs) and differentiated them into various vascular cell derivatives to study how NF-κB modulates human vascular cells under basal and inflammatory conditions. Multi-dimensional phenotypic assessments and transcriptomic analyses revealed that RelA deficiency affected vascular cells via modulating inflammation, survival, vasculogenesis, cell differentiation and extracellular matrix organization in a cell type-specific manner under basal condition, and that RelA protected vascular cells against apoptosis and modulated vascular inflammatory response upon tumor necrosis factor α (TNFα) stimulation. Lastly, further evaluation of gene expression patterns in IκBα knockout vascular cells demonstrated that IκBα acted largely independent of RelA signaling. Taken together, our data reveal a protective role of NF-κB/RelA in modulating human blood vessel homeostasis and map the human vascular transcriptomic landscapes for the discovery of novel therapeutic targets.

A NOVEL REGULATORY EFFECT OF AT1 RECEPTOR-INTERACTING MOLECULE ON VASCULAR SMOOTH MUSCLE CELLS

Objective: Previous studies reported that the production of superoxide is modulated by many factors including Ang II - AT1 receptor signaling. One of the major sources of superoxide in the aorta is NADPH oxidase located in the smooth muscle cells. The NADPH oxidase complex in the vascular smooth muscle cells consists of p22phox, Rac1, and Nox1. Previous studies showed that the carboxy-terminal cytoplasmic domain of AT1 receptor is involved in the control of receptor internalization and in linking receptor-mediated signal transduction to the specific biological response. Design and method: We previousy cloned a novel molecule interacting with carboxy-terminal domain of AT1 receptor, which we named ATRAP (for AT1 receptor-associated protein), using the yeast two-hybrid strategy. In this study, we tested the hypothesis that vascular smooth muscle cells express ATRAP and that ATRAP modulates Ang II-induced proliferative activity and oxidative stress in vascular smooth muscle cells. We identified that the ATRAP mRNA and protein were endogeneously expressed in VSMC, and found a colocalization of ATRAP and AT1 receptor in Ang II-stimulated VSMC. Results: The results of gain-of-function studies by adenoviral gene transfer demonstrated that overexpression of ATRAP significantly inhibited Ang II-mediated increases in c-fos gene transcription, BrdU incorpoaration, and mRNAs expression of NADPH oxidase complex (p < 0.05, n = 6). Conclusions: These results indicate that ATRAP significantly attenuates Ang II-mediated proliferative activity and oxidative stress in vascular smooth muscle cells, and may suggest a novel strategy to inhibit cardiovascular disease such as arteriosclerosis and hypertension.

Naked physically synthesized gold nanoparticles affect migration, mitochondrial activity, and proliferation of vascular smooth muscle cells.

IntroductionVascular smooth muscle cells (VSMCs) play an important role in the development and progression of atherosclerosis and vascular injuries in terms of proliferation and migration. Therefore, the aim of this study was to investigate the anti-migratory and proliferative effects of naked gold nanoparticles (AuNPs) on VSMCs.Materials and methodsOne set of physically synthesized AuNPs (pAuNPs) and three sets of chemically synthesized AuNPs (cAuNPs) were tested.Results and discussionAmong them, the pAuNPs were found to significantly and markedly inhibit platelet-derived growth factor (PDGF)-induced VSMC migration. Transmission electron microscopy revealed that the pAuNPs were ingested and aggregated in the cytoplasm at an early stage of treatment, while the viability of VSMCs was not affected within 24 hours of treatment. The pAuNP treatment enhanced cellular mitochondrial activity but inhibited basal and PDGF-induced VSMC proliferation, as determined by MTT, WST-1, and BrdU cell proliferation assays. Furthermore, the pAuNPs did not interfere with PDGF signaling or matrix metalloproteinase-2 expression/activity. Unlike the cAuNPs, the pAuNPs could markedly reduce VSMC adhesion to collagen, which was supported by the findings that the pAuNPs could inhibit collagen-induced tyrosine protein and focal adhesion kinase (FAK) phosphorylation and actin cytoskeleton reorganization during cell adhesion. The in vitro effects of the pAuNPs were confirmed in the in vivo rat balloon-injured carotid artery model by diminishing the proliferating VSMCs.ConclusionTaken together, the present study provides the first evidence that naked pAuNPs can reduce VSMC migration and compromise cell adhesion by affecting FAK and tyrosine-protein activation. The pAuNPs also have an inhibitory effect on PDGF-induced VSMC proliferation and can reduce proliferating/migrating VSMC expression in vivo.

Angiotensin II Activates the Expression of Inflammatory Markers and the Migration of Inflammatory Cells into the Aorta of Normotensive Mice

IntroductionAngiotensin II exerts important physiological functions on cardiovascular system homeostasis and may mediate actions leading to inflammatory responses. These actions lead to the activation of vascular smooth muscle cells (CMLVs) as well as the endothelium, in order to produce reactive oxygen species, inflammatory cytokines, chemokines and adhesion molecules inducing diapedesis of cells of myeloid origin. In this context, variations in Angiotensin II concentrations could occur in the body, leading to discrete increases that do not directly and immediately interfere with blood pressure but could stimulate the recruitment and activation of myeloid cells capable of initiating local inflammatory responses. Such a situation would provide important information for triggering a chronic process on the mechanisms of maintaining blood pressure and potentially leading to cardiovascular disease. This study aimed to verify the ability of angiotensin II to induce an inflammatory response in the aorta and if there is a relation with variations of arterial pressure, even if discrete.MethodsFor this, C57Bl/6 mice treated with saline solution (0.9%, control group) or Angiotensin II (30ng, Ang II group) were used. The animals were cannulated in the carotid artery and jugular vein, and 48 hours later PA and HR levels were recorded at baseline and after administration of saline or Ang II at 30 min, 1, 2, 6, 12 and 24 h. The evaluation of the baroreflex sensitivity was performed after administration of phenylephrine and sodium nitroprusside (100 and 150 ng). The evaluation of the inflammatory reaction in the aorta was performed by immunohistochemistry, using IL1‐β, TGF‐β, iNOS as inflammatory markers and CD45 as a marker of macrophages. The evaluation of α‐actin was performed in order to demonstrate a possible change in CMLV phenotype.ResultsAt the end of the treatments, we verified that there was no change in blood pressure or heart rate. In addition, the treatment did not alter the baroreflex sensitivity. A biphasic response was observed both for IL1‐β and TGF‐β expression and also for the presence of CD45 positive cells, with an acute increase (between 30 and 60 minutes) and another more chronic increase, between 24 and 48 hours. Positive staining for iNOS increased in the earlier period (30 minutes) in perivascular adipose tissue and in a longer period (24 hours) in tunica adventitia. Immunoblotting to α‐actin showed no alterations, suggesting that the applied dose of angiotensin II does not alter the aortic CMLV phenotype.ConclusionThe results suggest that angiotensin II, even at doses that do not alter blood pressure, is capable of inducing the expression of inflammatory markers and the migration of inflammatory cells into the aorta of normotensive mice. Thus, angiotensin II may be considered to be capable of increasing the propensity to develop a cardiovascular injury, even in normotensive individuals.Support or Funding InformationThis research was funded by Coordenação de Apoio a Pesquisa (CAPES) and São Paulo's state research funding agency Foundation (FAPESP).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Optogenetic stimulation of pericytes lacking alpha smooth muscle actin produces a decrease in capillary blood flow in the living mouse brain

Impaired cerebral blood flow contributes to the pathogenesis of numerous neurological diseases, ranging from dementia to traumatic brain injury. It is critical that we understand the cell types that control cerebral blood flow, so that we can target them to correct blood flow impairments and improve the trajectory of these diseases. Although it is accepted that vascular smooth muscle cells encircling arterioles can control blood flow, it is debated whether pericytes, which collectively adorn over 90% of the brain's vascular length, can also influence blood flow. To clarify the ability of pericytes to influence blood flow, we stimulated channelrhodopsin (ChR2) in individual pericytes using two photon illumination, as done previously (Hill, et al. Neuron 2015). Although we used mice that express ChR2 in both smooth muscle cells and pericytes, the spatial specificity of two photon excitation permitted selective optogenetic stimulation of individual pericytes, thus avoiding incidental activation of nearby vascular smooth muscle cells that also express ChR2. Simultaneously, with the same two photon laser used for excitation of ChR2 in pericytes, we measured capillary diameter and red blood cell velocity (RBCV) using intravenous dextrans. We found that pericyte excitation for 60 seconds produced a ~20% reduction in diameter and RBCV, indicating that pericytes can indeed influence blood flow. Surprisingly, the magnitude and kinetics of the decreases in diameter and RBCV were not different between pericytes with and without expression of alpha smooth muscle actin, a protein believed to be required for vascular contraction and blood flow control. Importantly, identical stimulation parameters did not produce hemodynamic changes in control mice with cytosolic YFP or membrane-bound GFP in smooth muscle cells and pericytes, indicating that our observed reductions in blood flow in ChR2 mice were not an artifactual result of light illumination, animal movement, or tissue damage. Further, the observed decrease in velocity and diameter in response to pericyte ChR2 stimulation was inhibited when we applied fasudil, a Rho kinase inhibitor and clinically-used vasodilator, to an intact dura (dose of 10 mM). Our results to date suggest that pericytes, even those without alpha smooth muscle actin, have the capacity to modulate blood flow. These findings will advance our understanding of vascular biology, and could be relevant for treating disease processes associated with deficits in capillary blood flow, such as dementia and traumatic brain injury. Support or Funding Information A.Y.S. thanks his support: NS085402, NS096997, NS097775, P20GM12345, NSF 1539034, AHA 14GRNT20480366, Alzheimer's Association, and CCAD. D.A.H. thanks his support: UL1 TR001450, TL1 TR001451, and F30NS096868. We optogenetically stimulated individual pericytes in vivo using two photon microscopy (A), and observed a significant decrease in the diameter and red blood cell flow in the underlying capillary (B). Surprisingly, similar responses were seen between pericytes with and without expression of alpha smooth muscle actin, an important protein in canonical vascular contraction mechanisms. Pericytes can therefore modulate blood flow using non-canonical mechanisms that can be targeted for therapies. This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Pharmacological inhibition of TRPV4 channels protects against early renal hypoperfusion induced by ischemia‐reperfusion in neonatal pigs

Renal ischemia‐reperfusion (IR) remains a significant cause of acute kidney injury (AKI) in neonates. Early renal vasoconstriction in ischemic AKI induces hypoperfusion and a rapid decline in kidney function. Mechanisms that underlie post‐ischemic renal hypoperfusion are poorly understood, but possibilities include alterations in ion channel‐mediated renal vasoregulation. Here, we show that pharmacological activation of TRPV4 channels promotes renal vasoconstriction and hypoperfusion in neonatal pigs. Bilateral renal ischemia followed by short‐term reperfusion increased TRPV4 channel protein expression and activity in preglomerular microvessels and vascular smooth muscle cells (SMCs), respectively. Renal IR caused oliguria and elevated serum or urine levels of AKI biomarkers. Renal IR also increased renal vascular resistance (RVR) and attenuated renal blood flow and glomerular filtration rate in the pigs. However, TRPV4 channel blockers HC 067047 and RN 1734 mitigated renal IR‐induced oliguria, hypoperfusion, and alterations in AKI biomarkers. Furthermore, renal IR increased serum angiotensin II (ANG II) concentration, but not renal vascular expression of type 1 ANG II receptors in the pigs. TRPV4 channel blockade reduced ANG II‐induced renal vasoconstriction, RVR increase, and renal hypoperfusion in the pigs. Together, our data suggest that increased renal vascular SMC TRPV4 channel expression, ANG II‐induced TRPV4 activation, and subsequent increase in RVR contribute to hypoperfusion and kidney insufficiency elicited by renal IR in neonatal pigs. We propose that multimodal signaling by renal vascular SMC TRPV4 channels regulates neonatal renal hemodynamics in health and disease.Support or Funding InformationR01DK101668This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

PDGFRβ Induces Ribosome Biogenesis in Aortic Smooth Muscle Cells

IntroductionArterial fibrosis (AF) is an important determinant of cardiovascular health. AF results in a thickened arterial wall and loss of compliance which is a direct result of excessive extracellular matrix (ECM) accumulation. Platelet Derived Growth Factor Receptor β (PDGFRβ) activation in vascular smooth muscle cells (VSMCs) results in AF that manifests with increased VSMC proliferation and increased collagen synthesis and deposition in the arteries. However, the mechanisms by which PDGFRβ activation results in AF are not fully elucidated.ObjectiveWe sought to investigate whether PDGFRβ regulates ribosome biogenesis (RB), and the extent to which increased RB downstream of PDGFRβ activation in VSMC mediates the AF‐associated structural and functional changes in mouse aorta.MethodsWe used Pdgfrb+/D849V mice (designated Bk) as a genetic model for AF where VSMC specific SM22Cre is used to drive expression of a conditional Pdgfrb knock‐in allele that encodes a constitutively active PDGFRβ in VSMC. In order to reduce ribosome content we crossed Bk mice with Rpl24Bst mice with a hypomorphic Rpl24 allele resulting in a 40% reduction in Ribosomal Protein L24. Arterial structure was evaluated in tissue sections. Ultrasound imaging was used to measure structural and functional arterial changes. We also serum‐starved rat aortic smooth muscle (A7r5) and 3T3 fibroblast cell lines and then stimulated with PDGF‐BB. RB was evaluated by measuring ribosomal proteins using Western Blot and assessing rRNA as RNA/DNA ratio using a fluorometric assay.ResultsCompared to wild type, Bk aortas were significantly larger with increased diameter and wall thickness and were significantly stiffer with reduced distension. Secondary to the arterial changes, Bk mice showed decreased cardiac contractility at 6 months of age that was not apparent at 3 months, but cardiac output did not decrease. Stimulating serum‐starved 3T3 fibroblasts and A7r5 smooth muscle cell lines with PDGF‐BB resulted in RB measured as increased ribosomal proteins and RNA/DNA ratio. In Bk mice, VSMC consisted of significantly more ribosomal proteins (RPL26, RPL10A, RPL17, RPS3 and RPS6, p&gt;0.05) and increased transcript and RNA/DNA ratio (150%, p&gt;0.05). Altering ribosomal proteins using Rpl24Bst partially attenuated some of these changes.ConclusionsThis study highlights the novel concept that RB is an important factor in AF. We found that RB and ribosome content is increased in VSMC in response to PDGFRβ activation and that altering ribosomes using Rpl24Bst mutation attenuated the arterial changes associated with PDGFRβ activation. It can be expected that RB will prove to be instrumental for AF in VSMC, however a few unanswered questions still remain. First, what are the downstream signaling events by which PDGFRβ stimulates RB? And second, what are the mechanisms by which increased ribosome content results in increased vascular ECM deposition? In my future research, I will investigate the signaling pathways downstream of PDGFRβ that result in RB. I will also identify specific transcripts that are targeted by ribosomes in response to PDGFRβ activation and will test the efficacy of ribosome‐modulating interventions in inhibiting PDGFRβ‐induced AF.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The intersection of protein disulfide isomerase and cancer associated thrombosis

The mechanisms underlying the hypercoagulability of cancer are complex and include the upregulation coagulation factors or procoagulant proteins, shedding of microparticles, and direct activation of vascular cells. Protein disulfide isomerase (PDI) is a thiol isomerase secreted from activated platelets and endothelial cells and plays a critical role in both platelet aggregation and fibrin generation. A number of potential intravascular targets of PDI have been identified including cell surface receptors (e.g. β-integrins and glycoprotein Ib), receptor ligands (e.g. fibrinogen and von Willebrand factor), serine proteases (e.g. cathepsin G and kallekrein-14), and coagulation factors (e.g. factor XI and factor V). Recent clinical studies demonstrated that a small molecule inhibitor of PDI, isoquercetin, decreases platelet-dependent thrombin generation and PDI activity in plasma following oral administration. This review explores the mechanistic overlap between the molecular drivers of cancer associated thrombosis and the potential roles PDI plays in mediating thrombosis. These molecular insights provide rationale for clinical trials targeting PDI to prevent thrombosis in cancer patients.