Association between the Hypomethylation of Osteopontin and Integrin β3 Promoters and Vascular Smooth Muscle Cell Phenotype Switching in Great Saphenous Varicose Veins

Lower extremity varicose veins are a common condition in vascular surgery and proliferation of vascular smooth muscle cells (VSMCs) in the intima is a significant pathological feature of varicosity. However, the pathogenesis of varicose veins is not fully understood. Osteopontin (OPN) could promote the migration and adhesion of VSMCs through the cell surface receptor integrin β3 and the cooperation of OPN and integrin β3 is involved in many vascular diseases. However, the role of OPN and integrin β3 in varicosity remains unclear. In the current study, we found that the methylation levels in the promoter regions of OPN and integrin β3 genes in the VSMCs of varicose veins are reduced and the protein expression of OPN and integrin β3 are increased, compared with normal veins. Furthermore, it was observed that VSMCs in the neointima of varicose veins were transformed into the synthetic phenotype. Collectively, hypomethylation of the promoter regions for OPN and integrin β3 genes may increase the expression of these genes in varicosity, which is closely related to VSMC phenotype switching. Hypomethylation of the promoter regions for OPN and integrin β3 genes may be a key factor in the pathogenesis of varicosity.     

CCN2 Increases TGF-β Receptor Type II Expression in Vascular Smooth Muscle Cells: Essential Role of CCN2 in the TGF-β Pathway Regulation

The cellular communication network factor 2 (CCN2/CTGF) has been traditionally described as a mediator of the fibrotic responses induced by other factors including the transforming growth factor β (TGF-β). However, several studies have defined a direct role of CCN2 acting as a growth factor inducing oxidative and proinflammatory responses. The presence of CCN2 and TGF-β together in the cellular context has been described as a requisite to induce a persistent fibrotic response, but the precise mechanisms implicated in this relation are not described yet. Considering the main role of TGF-β receptors (TβR) in the TGF-β pathway activation, our aim was to investigate the effects of CCN2 in the regulation of TβRI and TβRII levels in vascular smooth muscle cells (VSMCs). While no differences were observed in TβRI levels, an increase in TβRII expression at both gene and protein level were found 48 h after stimulation with the C-terminal fragment of CCN2 (CCN2(IV)). Cell pretreatment with a TβRI inhibitor did not modify TβRII increment induced by CCN2(VI), demonstrating a TGF-β-independent response. Secondly, CCN2(IV) rapidly activated the SMAD pathway in VSMCs, this being crucial in the upregulation of TβRII since the preincubation with an SMAD3 inhibitor prevented it. Similarly, pretreatment with the epidermal growth factor receptor (EGFR) inhibitor erlotinib abolished TβRII upregulation, indicating the participation of this receptor in the observed responses. Our findings suggest a direct role of CCN2 maintaining the TGF-β pathway activation by increasing TβRII expression in an EGFR-SMAD dependent manner activation.     

Yohimbine,an α2-Adrenoceptor Antagonist,Suppresses PDGF-BB-Stimulated Vascular Smooth Muscle Cell Proliferation by Downregulating the PLCγ1 Signaling Pathway

Yohimbine (YOH) has antiproliferative effects against breast cancer and pancreatic cancer; however, its effects on vascular proliferative diseases such as atherosclerosis remain unknown. Accordingly, we investigated the inhibitory mechanisms of YOH in vascular smooth muscle cells (VSMCs) stimulated by platelet-derived growth factor (PDGF)-BB, a major mitogenic factor in vascular diseases. YOH (5–20 μM) suppressed PDGF-BB-stimulated a mouse VSMC line (MOVAS-1 cell) proliferation without inducing cytotoxicity. YOH also exhibited antimigratory effects and downregulated matrix metalloproteinase-2 and -9 expression in PDGF-BB-stimulated MOVAS-1 cells. It also promoted cell cycle arrest in the initial gap/first gap phase by upregulating p27Kip1 and p53 expression and reducing cyclin-dependent kinase 2 and proliferating cell nuclear antigen expression. We noted phospholipase C-γ1 (PLCγ1) but not ERK1/2, AKT, or p38 kinase phosphorylation attenuation in YOH-modulated PDGF-BB-propagated signaling pathways in the MOVAS-1 cells. Furthermore, YOH still inhibited PDGF-BB-induced cell proliferation and PLCγ1 phosphorylation in MOVAS-1 cells with α2B-adrenergic receptor knockdown. YOH (5 and 10 mg/kg) substantially suppressed neointimal hyperplasia in mice subjected to CCA ligation for 21 days. Overall, our results reveal that YOH attenuates PDGF-BB-stimulated VSMC proliferation and migration by downregulating a α2B-adrenergic receptor–independent PLCγ1 pathway and reduces neointimal formation in vivo. Therefore, YOH has potential for repurposing for treating atherosclerosis and other vascular proliferative diseases.     

Evodiamine Attenuates PDGF-BB-Induced Migration of Rat Vascular Smooth Muscle Cells through Activating PPARγ

The uncontrolled migration of vascular smooth muscle cells (VSMCs) into the intima is a critical process in the development of atherosclerosis. Evodiamine, an indole alkaloid extracted from the Chinese medicine evodia, has been shown to inhibit tumor cell invasion and protect the cardiovascular system, but its effects on VSMCs remain unknown. In the present study, we investigated the inhibitory effects of evodiamine on the platelet-derived growth factor-BB (PDGF-BB)-induced VSMC migration using wound healing and transwell assays, and assessed its role in decreasing the protein levels of matrix metalloproteinases and cell adhesion molecules. More importantly, we found that evodiamine activated the expression and nuclear translocation of peroxisome proliferator-activated receptor γ (PPARγ). Inhibition of PPARγ activity by using its antagonist T0070907 and its specific siRNA oligonucleotides significantly attenuated the inhibitory effects of evodiamine on VSMC migration. Taken together, our results indicate a promising anti-atherogenic effect of evodiamine through attenuation of VSMC migration by activating PPARγ.     

Adenylate Kinase 4—A Key Regulator of Proliferation and Metabolic Shift in Human Pulmonary Arterial Smooth Muscle Cells via Akt and HIF-1α Signaling Pathways

Increased proliferation of pulmonary arterial smooth muscle cells (PASMCs) in response to chronic hypoxia contributes to pulmonary vascular remodeling in pulmonary hypertension (PH). PH shares numerous similarities with cancer, including a metabolic shift towards glycolysis. In lung cancer, adenylate kinase 4 (AK4) promotes metabolic reprogramming and metastasis. Against this background, we show that AK4 regulates cell proliferation and energy metabolism of primary human PASMCs. We demonstrate that chronic hypoxia upregulates AK4 in PASMCs in a hypoxia-inducible factor-1α (HIF-1α)-dependent manner. RNA interference of AK4 decreases the viability and proliferation of PASMCs under both normoxia and chronic hypoxia. AK4 silencing in PASMCs augments mitochondrial respiration and reduces glycolytic metabolism. The observed effects are associated with reduced levels of phosphorylated protein kinase B (Akt) as well as HIF-1α, indicating the existence of an AK4-HIF-1α feedforward loop in hypoxic PASMCs. Finally, we show that AK4 levels are elevated in pulmonary vessels from patients with idiopathic pulmonary arterial hypertension (IPAH), and AK4 silencing decreases glycolytic metabolism of IPAH-PASMCs. We conclude that AK4 is a new metabolic regulator in PASMCs interacting with HIF-1α and Akt signaling pathways to drive the pro-proliferative and glycolytic phenotype of PH.     

Increase in Epithelial Permeability and Cell Metabolism by High Mobility Group Box 1, Inflammatory Cytokines and TPEN in Caco-2 Cells as a Novel Model of Inflammatory Bowel Disease

High mobility group box 1 protein (HMGB1) is involved in the pathogenesis of inflammatory bowel disease (IBD). Patients with IBD develop zinc deficiency. However, the detailed roles of HMGB1 and zinc deficiency in the intestinal epithelial barrier and cellular metabolism of IBD remain unknown. In the present study, Caco-2 cells in 2D culture and 2.5D Matrigel culture were pretreated with transforming growth factor-β (TGF-β) type 1 receptor kinase inhibitor EW-7197, epidermal growth factor receptor (EGFR) kinase inhibitor AG-1478 and a TNFα antibody before treatment with HMGB1 and inflammatory cytokines (TNFα and IFNγ). EW-7197, AG-1478 and the TNFα antibody prevented hyperpermeability induced by HMGB1 and inflammatory cytokines in 2.5D culture. HMGB1 affected cilia formation in 2.5D culture. EW-7197, AG-1478 and the TNFα antibody prevented the increase in cell metabolism induced by HMGB1 and inflammatory cytokines in 2D culture. Furthermore, ZnSO₄ prevented the hyperpermeability induced by zinc chelator TPEN in 2.5D culture. ZnSO₄ and TPEN induced cellular metabolism in 2D culture. The disruption of the epithelial barrier induced by HMGB1 and inflammatory cytokines contributed to TGF-β/EGF signaling in Caco-2 cells. The TNFα antibody and ZnSO₄ as well as EW-7197 and AG-1478 may have potential for use in therapy for IBD.     

Melatonin Suppresses Hypoxia-Induced Migration of HUVECs via Inhibition of ERK/Rac1 Activation

Melatonin, a naturally-occurring hormone, possesses antioxidant properties and ameliorates vascular endothelial dysfunction. In this study, we evaluate the impact of melatonin on the migratory capability of human umbilical vein endothelial cells (HUVECs) to hypoxia and further investigate whether ERK/Rac1 signaling is involved in this process. Here, we found that melatonin inhibited hypoxia-stimulated hypoxia-inducible factor-1α (HIF-1α) expression and cell migration in a dose-dependent manner. Mechanistically, melatonin inhibited Rac1 activation and suppressed the co-localized Rac1 and F-actin on the membrane of HUVECs under hypoxic condition. In addition, the blockade of Rac1 activation with ectopic expression of an inactive mutant form of Rac1-T17N suppressed HIF-1α expression and cell migration in response to hypoxia, as well, but constitutive activation of Rac1 mutant Rac1-V12 restored HIF-1α expression, preventing the inhibition of melatonin on cell migration. Furthermore, the anti-Rac1 effect of melatonin in HUVECs appeared to be associated with its inhibition of ERK phosphorylation, but not that of the PI3k/Akt signaling pathway. Taken together, our work indicates that melatonin exerts an anti-migratory effect on hypoxic HUVECs by blocking ERK/Rac1 activation and subsequent HIF-1α upregulation.     

Impact of an Altered Wnt1/β-Catenin Expression on Clinicopathology and Prognosis in Clear Cell Renal Cell Carcinoma

In renal cell carcinoma (RCC), single members of the Wnt/β-catenin signaling cascade were recently identified to contribute to cancer progression. However, the role of Wnt1, one of the key ligands in β-catenin regulation, is currently unknown in RCC. Therefore, alterations of the Wnt1/β-catenin axis in clear cell RCC (ccRCC) were examined with regard to clinicopathology, overall survival (OS) and cancer specific survival (CSS). Corresponding ccRCCs and benign renal tissue were analyzed in 278 patients for Wnt1 and β-catenin expression by immunohistochemistry in tissue microarrays. Expression scores, including intensity and percentage of stained cells, were compared between normal kidney and ccRCCs. Data was categorized according to mean expression scores and correlated to tumor and patients’ characteristics. Survival was analyzed according to the Kaplan-Meier and log-rank test. Univariable and multivariable Cox proportional hazard regression models were used to explore the independent prognostic value of Wnt1 and β-catenin. In ccRCCs, high Wnt1 was associated with increased tumor diameter, stage and vascular invasion (p ≤ 0.02). High membranous β-catenin was associated with advanced stage, vascular invasion and tumor necrosis (p ≤ 0.01). Higher diameter, stage, node involvement, grade, vascular invasion and sarcomatoid differentiation (p ≤ 0.01) were found in patients with high cytoplasmic β-catenin. Patients with a high cytoplasmic β-catenin had a significantly reduced OS (hazard ratio (HR) 1.75) and CSS (HR 2.26), which was not independently associated with OS and CSS after adjustment in the multivariable model. Increased ccRCC aggressiveness was reflected by an altered Wnt1/β-catenin signaling. Cytoplasmic β-catenin was identified as the most promising candidate associated with unfavorable clinicopathology and impaired survival. Nevertheless, the shift of membranous β-catenin to the cytoplasm with a subsequently increased nuclear expression, as shown for other malignancies, could not be demonstrated to be present in ccRCC.     

The Neurovascular Unit Dysfunction in Alzheimer’s Disease

Alzheimer’s disease (AD) is the most common neurodegenerative disease worldwide. Histopathologically, AD presents with two hallmarks: neurofibrillary tangles (NFTs), and aggregates of amyloid β peptide (Aβ) both in the brain parenchyma as neuritic plaques, and around blood vessels as cerebral amyloid angiopathy (CAA). According to the vascular hypothesis of AD, vascular risk factors can result in dysregulation of the neurovascular unit (NVU) and hypoxia. Hypoxia may reduce Aβ clearance from the brain and increase its production, leading to both parenchymal and vascular accumulation of Aβ. An increase in Aβ amplifies neuronal dysfunction, NFT formation, and accelerates neurodegeneration, resulting in dementia. In recent decades, therapeutic approaches have attempted to decrease the levels of abnormal Aβ or tau levels in the AD brain. However, several of these approaches have either been associated with an inappropriate immune response triggering inflammation, or have failed to improve cognition. Here, we review the pathogenesis and potential therapeutic targets associated with dysfunction of the NVU in AD.     

NLRP3 Inflammasome Activation Controls Vascular Smooth Muscle Cells Phenotypic Switch in Atherosclerosis

(1) Background: Monocytes and nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3) inflammasome orchestrate lipid-driven amplification of vascular inflammation promoting the disruption of the fibrous cap. The components of the NLRP3 inflammasome are expressed in macrophages and foam cells within human carotid atherosclerotic plaques and VSMCs in hypertension. Whether monocytes and NLRP3 inflammasome activation are direct triggers of VSMC phenotypic switch and plaque disruption need to be investigated. (2) Methods: The direct effect of oxLDL-activated monocytes in VSMCs co-cultured system was demonstrated via flow cytometry, qPCR, ELISA, caspase 1, and pyroptosis assay. Aortic roots of VSMCs lineage tracing mice fed normal or high cholesterol diet and human atherosclerotic plaques were used for immunofluorescence quantification of NLRP3 inflammasome activation/VSMCs phenotypic switch. (3) Results: OxLDL-activated monocytes reduced α-SMA, SM22α, Oct-4, and upregulation of KLF-4 and macrophage markers MAC2, F4/80 and CD68 expression as well as caspase 1 activation, IL-1β secretion, and pyroptosis in VSMCs. Increased caspase 1 and IL-1β in phenotypically modified VSMCs was detected in the aortic roots of VSMCs lineage tracing mice fed high cholesterol diet and in human atherosclerotic plaques from carotid artery disease patients who experienced a stroke. (4) Conclusions: Taken together, these results provide evidence that monocyte promote VSMC phenotypic switch through VSMC NLRP3 inflammasome activation with a likely detrimental role in atherosclerotic plaque stability in human atherosclerosis.     

The Effects of CoCl2 on HIF-1α Protein under Experimental Conditions of Autoprogressive Hypoxia Using Mouse Models

It is well known that cobalt chloride (CoCl₂) can enhance the stability of hypoxia-inducible factor (HIF)-1α. The aim of this study is to detect the effect of CoCl₂ on the hypoxia tolerance of mice which were repeatedly exposed to autoprogressive hypoxia. Balb/c mice were randomly divided into groups of chemical pretreatment and normal saline (NS), respectively injected with CoCl₂ and NS 3 h before exposure to hypoxia for 0 run (H0), 1 run (H1), and 4 runs (H4). Western Blot, electrophoretic mobility shift assay (EMSA), extracellular recordings population spikes in area cornus ammonis I (CA 1) of mouse hippocampal slices and real-time were used in this study. Our results demonstrated that the tolerance of mice to hypoxia, the changes of HIF-1α protein level and HIF-1 DNA binding activity in mice hippocampus, the mRNA level of erythropoietin (EPO) and vascular endothelial growth factor (VEGF), and the disappearance time of population spikes of hippocampal slices were substantially different between the control group and the CoCl₂ group. Over-induction of HIF-1α by pretreatment with CoCl₂ before hypoxia did not increase the hypoxia tolerance.     

Insoluble Vascular Amyloid Deposits Trigger Disruption of the Neurovascular Unit in Alzheimer’s Disease Brains

Alzheimer’s disease (AD) is a neurodegenerative disease, characterized histopathologically by intra-neuronal tau-related lesions and by the accumulation of amyloid β-peptide (Aβ) in the brain parenchyma and around cerebral blood vessels. According to the vascular hypothesis of AD, an alteration in the neurovascular unit (NVU) could lead to Aβ vascular accumulation and promote neuronal dysfunction, accelerating neurodegeneration and dementia. To date, the effects of insoluble vascular Aβ deposits on the NVU and the blood–brain barrier (BBB) are unknown. In this study, we analyze different Aβ species and their association with the cells that make up the NVU. We evaluated post-mortem AD brain tissue. Multiple immunofluorescence assays were performed against different species of Aβ and the main elements that constitute the NVU. Our results showed that there are insoluble vascular deposits of both full-length and truncated Aβ species. Besides, insoluble aggregates are associated with a decrease in the phenotype of the cellular components that constitute the NVU and with BBB disruption. This approach could help identify new therapeutic targets against key molecules and receptors in the NVU that can prevent the accumulation of vascular fibrillar Aβ in AD.     

4-Hydroxyphenylacetic Acid Attenuated Inflammation and Edema via Suppressing HIF-1α in Seawater Aspiration-Induced Lung Injury in Rats

4-Hydroxyphenylacetic acid (4-HPA) is an active component of Chinese herb Aster tataricus which had been widely used in China for the treatment of pulmonary diseases. The aim of this study is to investigate the effect of 4-HPA on seawater aspiration-induced lung injury. Pulmonary inflammation and edema were assessed by enzyme-linked immunosorbent assay (ELISA), bronchoalveolar lavage fluid (BALF) white cell count, Evans blue dye analysis, wet to dry weight ratios, and histology study. Hypoxia-inducible factor-1α (HIF-1α) siRNA and permeability assay were used to study the effect of 4-HPA on the production of inflammatory cytokines and monolayer permeability in vitro. The results showed that 4-HPA reduced seawater instillation-induced mortality in rats. In lung tissues, 4-HPA attenuated hypoxia, inflammation, vascular leak, and edema, and decreased HIF-1α protein level. In primary rat alveolar epithelial cells (AEC), 4-HPA decreased hypertonicity- and hypoxia-induced HIF-1α protein levels through inhibiting the activations of protein translational regulators and via promoting HIF-1α protein degradation. In addition, 4-HPA lowered inflammatory cytokines levels through suppressing hypertonicity- and hypoxia-induced HIF-1α in NR8383 macrophages. Moreover, 4-HPA decreased monolayer permeability through suppressing hypertonicity and hypoxia-induced HIF-1α, which was mediated by inhibiting vascular endothelial growth factor (VEGF) in rat lung microvascular endothelial cell line (RLMVEC). In conclusion, 4-HPA attenuated inflammation and edema through suppressing hypertonic and hypoxic induction of HIF-1α in seawater aspiration-induced lung injury in rats.     

Paricalcitol Improves Hypoxia-Induced and TGF-β1-Induced Injury in Kidney Pericytes

Recently, the role of kidney pericytes in kidney fibrosis has been investigated. This study aims to evaluate the effect of paricalcitol on hypoxia-induced and TGF-β1-induced injury in kidney pericytes. The primary cultured pericytes were pretreated with paricalcitol (20 ng/mL) for 90 min before inducing injury, and then they were exposed to TGF-β1 (5 ng/mL) or hypoxia (1% O₂ and 5% CO₂). TGF-β1 increased α-SMA and other fibrosis markers but reduced PDGFRβ expression in pericytes, whereas paricalcitol reversed the changes. Paricalcitol inhibited the TGF-β1-induced cell migration of pericytes. Hypoxia increased TGF-β1, α-SMA and other fibrosis markers but reduced PDGFRβ expression in pericyte, whereas paricalcitol reversed them. Hypoxia activated the HIF-1α and downstream molecules including prolyl hydroxylase 3 and glucose transporter-1, whereas paricalcitol attenuated the activation of the HIF-1α-dependent molecules and TGF-β1/Smad signaling pathways in hypoxic pericytes. The gene silencing of HIF-1α vanished the hypoxia-induced TGF-β1, α-SMA upregulation, and PDGFRβ downregulation. The effect of paricalcitol on the HIF-1α-dependent changes of fibrosis markers was not significant after the gene silencing of HIF-1α. In addition, hypoxia aggravated the oxidative stress in pericytes, whereas paricalcitol reversed the oxidative stress by increasing the antioxidant enzymes in an HIF-1α-independent manner. In conclusion, paricalcitol improved the phenotype changes of pericyte to myofibroblast in TGF-β1-stimulated pericytes. In addition, paricalcitol improved the expression of fibrosis markers in hypoxia-exposed pericytes both in an HIF-1α-dependent and independent manner.