Blood mononuclear cells induce accelerated vascular remodeling under acute inflammation in vitro

Background: Mononuclear cells play a crucial role in vascular remodelling and cardiovascular disease. Monocytes are recruited to sites of collateral growth where they contribute to arteriogenesis. However, prolonged presence or increased accumulation of these cells can compromise tissue integrity and lead to atherosclerosis. Bone morphogenetic proteins (BMPs) are members of the Transforming growth factor (TGF)-β family of cytokines and play important roles in vascular remodelling and function. It was shown that BMP-2 is up-regulated in calcified atherosclerotic plaques. BMP-2 contributes to calcification and inflammation of the vascular wall. It is unknown whether these effects of BMP-2 are also due to its effects on monocyte function or/and on the monocyte-endothelial cell interactions. Methods: Human monocytes were isolated from peripheral blood through gradient centrifugation and subsequent negative immunological magnet isolation. Monocyte motility was analysed by the modified Boyden chamber migration assay. Protein phosphorylation was assessed by immunoblotting. For endothelial cell adhesion assays, mouse endothelial cells were stimulated with different ligands and mononuclear cell adhesion was analysed. The involvement of different signalling cascades was analysed using specific kinase inhibitors. Results: Here we show that stimulation of primary monocytes with BMP-2 results in monocyte polarization. BMP-2 stimulated monocyte chemotaxis in a dose-dependent manner, while addition of Noggin, a natural BMP-2/4/7 antagonist, hindered monocyte chemotaxis towards BMP-2. Additionally BMP2 induces mononuclear cell adhesion on fibronectin. BMPs signal into the cells by activating both Smad and non-Smad signalling pathways such as PI3K and p38 MAPK pathway. Our results suggest that both PI3K and p38 pathways are involved in BMP-2-induced monocyte migration. Moreover, BMP-2 stimulated monocyte adhesion on endothelial cells by inducing inflammatory responses in endothelial cells in a PI3K and p38 dependent manner. Conclusions: Our results reveal that BMP-2 is a novel stimulator of human monocyte function and suggest that BMP-2 may exert its pro-inflammatory effects and pro-calcification action in atherosclerosis by activating the endothelium and in addition by recruitment of monocytes.

BACKGROUNDPulmonary hypoplasia accompanied by pulmonary hypertension resistant to treatment is an important feature of congenital diaphragmatic hernia (CDH). The pathogenesis of the pulmonary vascular abnormalities in CDH remains to be...

<b>Abstract ID 56626</b> <b>Poster Board 156</b> Vascular remodeling is associated with target organ damage and fatal cardiovascular events. The molecular mechanisms that control vascular remodeling are still poorly understood, limiting effective therapeutic approaches. Vascular smooth muscle cells (SMCs), in part via activation of the Wnt/β-catenin signaling pathway, contribute importantly to vascular remodeling. The β-catenin C-terminal domain is required in SMCs for artery formation during embryogenesis, but its role in vascular remodeling in adulthood is unknown. Thus, the aim of this study was to define the importance of β-catenin C-terminus in SMCs during vascular remodeling and the underlying mechanisms. We found that mice expressing a C-terminus-deficient β-catenin in SMCs show reduced vascular remodeling and decreased SMC proliferation after arterial injury. In line with these findings, we observed that treatment with E7386, a novel β-catenin C-terminus inhibitor, reduces cell proliferation of both human and mouse vascular SMCs. RNA-seq analysis revealed a downregulation of the sphingosine-1-phosphate receptor 1 (S1pr1) transcript in β-catenin C-terminus-deficient SMCs. This receptor is known to be important in vascular remodeling, but its regulation is not fully elucidated. Interestingly, we found that β-catenin interacts with the S1pr1 promoter and acts through its C-terminal domain to activate S1pr1 transcription and protein expression, resulting in enhanced SMC proliferation. Consistent with these observations, S1PR1 expression was markedly decreased in SMCs of arteries from mice lacking the β-catenin C-terminus, and re-establishing S1PR1 expression in SMCs restored vascular remodeling in those mice. Taken together, these findings identify the β-catenin C-terminal output as a novel upstream regulator of S1PR1 expression and define a functional interaction between the canonical Wnt and sphingosine-1-phospate signaling pathways that drives vascular remodeling after injury. Our study suggests that targeting β-catenin C-terminus/S1PR1 axis could provide a therapeutic strategy to reduce vascular remodeling and cardiovascular disease. This study was supported by the American Heart Association Postdoctoral Fellowship 874619 (G.H.O-P), a Career Development Award from the American Heart Association 19CDA34660217 (D.R-B.), and NIH grant R01HL128066 (N.S.).

Although elevated levels of homocysteine (Hcy) known as hyperhomocysteinemia (HHcy) are associated with increased inflammation and vascular remodeling, the mechanism of Hcy-mediated inflammation and vascular remodeling is unclear. The matrix metalloproteinases (MMPs) and adhesion molecules play an important role in vascular remodeling. We hypothesized that HHcy induces inflammation by increasing adhesion molecules and matrix protein expression. Endothelial cells were supplemented with high methionine, and Hcy accumulation was measured by HPLC. Nitric oxide (NO) bioavailability was detected by a NO probe. The protein expression was measured by Western blot analysis. MMP-9 activity was detected by gelatin-gel zymography. We demonstrated that methionine supplement promoted upregulation of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) through increased Hcy accumulation. In addition, increased synthesis of collagen type-1 was also observed. MMP-9 gene expression and protein activity were increased in methionine supplement groups. 3-Deazaadenosine (DZA), an adenosine analogue, prevented high methionine-induced ICAM-1 and VCAM-1 expression and collagen type-1 synthesis. Transfection of endothelial cells with cystathionine-beta-synthase (CBS) gene construct, which converts Hcy to cystathionine, reduced Hcy accumulation in high methionine-fed cells. CBS gene transfection reduced the inflammatory response, as evident by attenuated ICAM-1 and VCAM-1 expression. Furthermore, collagen type-1 expression and MMP-9 activity were dramatically attenuated with CBS gene transfection. These results suggested that methionine supplement increased Hcy accumulation, which was associated with inflammatory response and matrix remodeling such as collagen type-1 synthesis and MMP-9 activity. However, in vitro DZA and CBS gene therapy successfully treated the HHcy-induced inflammatory reaction in the methionine metabolism pathway.

Vascular smooth muscle cells (SMCs), in part via activation of the Wnt/β‐catenin signaling pathway, contribute importantly to vascular remodeling. While the C‐terminal domain of β‐catenin links the pathway to gene expression, how this domain and related molecular mechanisms are involved in vascular remodeling is unknown. Inhibitors targeting specifically the C‐terminal domain of β‐catenin have been developed, but their use in obstructive vascular disease is not yet justified because of this gap of knowledge. Our group has previously shown that β‐catenin C‐terminal domain in SMCs is essential for arterial wall assembly during embryogenesis, but its role in vascular remodeling in adulthood is unknown. Therefore, the objective of this study was to define the importance of β‐catenin C‐terminal domain in SMCs during vascular remodeling and the underlying molecular mechanisms.To test the hypothesis that β‐catenin C‐terminal domain in SMCs is required for vascular remodeling, mice bearing a mutant β‐catenin allele (ΔC) that prevents the transcription of the C‐terminal domain were crossed with the SMC‐specific SMA‐CreERT2 line to generate mice bearing one β‐catenin flox allele and one knockin mutant (Ctnnb1ΔC/flox) or wt (Ctnnb1wt/flox) allele. In this system, tamoxifen administration removes the flox allele, resulting in mice bearing the mutant allele (SMβCΔC mice) or the wt allele (SMβCwt/‐ mice) as the only sources of β‐catenin in SMCs. To induce vascular remodeling, we performed carotid artery ligation one week after tamoxifen injection in male and female adult mice. Carotid arteries from both male and female SMβCΔC mice showed reduced neointima formation after injury induced by ligation compared to SMβCwt/‐ mice. Arteries from SMβCΔC mice also showed reduced SMC proliferation and increased SMC apoptosis and differentiation markers. We also isolated mouse aortic SMCs (MASMCs) from Ctnnb1ΔC/flox mice, and transduced these cells with GFP‐ or Cre‐expressing adenovirus to obtain control (βCControl) or cells expressing β‐cateninΔC(βCΔC) and found that βCΔC MASMCs show reduced growth compared to βCControl. To gain further insight on the mechanisms underlying the effects of β‐catenin C‐terminus, we performed whole‐transcriptome analysis by RNA‐seq of βCΔC and βCControl MASMCs and found a downregulation of the sphingosine‐1‐phosphate receptor 1 (S1pr1) gene in βCΔC MASMCs. This receptor is known to be important in vascular remodeling, but its regulation is not fully elucidated. Interestingly, we found decreased S1PR1 expression in SMCs of injured arteries from SMβC∆C mice. Consistent with this observation, βCΔC MASMCs showed reduced S1PR1 mRNA and protein levels and decreased S1pr1 promoter activity, which were all rescued by the transfection of a constitutively active form of β‐catenin. By using a S1PR1lox/stop/lox conditional gain‐of‐function mouse line, we found that overexpression of S1PR1 in SMCs rescues the injury‐induced neointima formation in SMβC∆C mice. Consistently, overexpression of S1PR1 in SMCs restored βCΔC MASMC growth towards βCControl levels. Taken together, our results define an essential SMC β‐catenin C‐terminal domain–S1PR1 axis that drives neointima formation after arterial injury, and identify multiple points for potential therapeutic intervention to control vascular remodeling in disease.

A role for muscle LIM protein (MLP) in vascular remodeling

It has been proposed that membrane microdomains, caveolae, in vascular cells are critical for signal transduction and downstream functions induced by angiotensin II (AngII). We have tested our hypothesis that caveolin-1 (Cav1), a major structural protein of vascular caveolae, plays a critical role in the development of vascular remodeling by AngII via regulation of epidermal growth factor receptor and vascular endothelial adhesion molecule-1. Cav1-/- and control Cav+/+ mice were infused with AngII for 2 weeks to induce vascular remodeling and hypertension. On AngII infusion, histological assessments demonstrated medial hypertrophy and perivascular fibrosis of aorta and coronary and renal arteries in Cav1+/+ mice compared with sham-operated Cav1+/+ mice. AngII-infused Cav1+/+ mice also showed a phenotype of cardiac hypertrophy with increased heart weight to body weight ratio compared with control Cav1+/+ mice. In contrast, Cav1-/- mice infused with AngII showed attenuation of vascular remodeling but not cardiac hypertrophy. Similar levels of AngII-induced hypertension were found in both Cav1+/+ and Cav1-/- mice as assessed by telemetry. In Cav1+/+ mice, AngII enhanced tyrosine-phosphorylated epidermal growth factor receptor staining in the aorta, which was attenuated in Cav1-/- mice infused with AngII. Enhanced Cav1 and vascular endothelial adhesion molecule-1 expression was also observed in aorta from AngII-infused Cav1+/+ mice but not in Cav1-/- aorta. Experiments with vascular cells further provided a potential mechanism for our in vivo findings. These data suggest that Cav1, and presumably caveolae, in vascular smooth muscle and the endothelium plays a critical role in vascular remodeling and inflammation independent of blood pressure or cardiac hypertrophy regulation.

Acute coronary syndrome (ACS) is the main manifestation of cardiovascular disease and the primary cause of adult hospitalization in China. There is an urgent demand for novel biomarkers for the diagnosis of ACS. Although plasma cysteine-rich protein 61 (Cyr61) has been previously reported to be accurate for ACS diagnosis, the accuracy of exosomal Cyr61 in ACS diagnosis remains unknown. In the present study, the aim was to assess the potential of applying exosomal Cyr61 in ACS diagnosis and to explore the role of Cyr61 in vascular remodeling in vitro. The abundance of Cyr61 in plasma-derived exosomes from patients with unstable angina pectoris (UAP), acute myocardial infarction (AMI) patients in addition to those isolated from healthy individuals were detected using an ELISA kit. The association between exosomal Cyr61 levels and clinical characteristics of ACS patients was analyzed through χ2 test, Fisher's exact test and Student's t-test. Receiver operating characteristic (ROC) curve analysis was used to determine the accuracy of using exosomal Cyr61 as a biomarker of ACS diagnosis. Furthermore, independent predictors of the existence of ACS were investigated through a multivariate analysis. Subsequently, the role of Cyr61 on vascular remodeling was evaluated in vascular smooth muscle cells (VSMCs) upon oxidized low-density lipoprotein (ox-LDL) treatment by performing Cyr61 knockdown, Cell Counting Kit-8, flow cytometry and Transwell assays. Exosomal Cyr61 expression was found to be significantly elevated in patients with ACS compared with that in healthy individuals. In addition, exosomal Cyr61 levels were associated with sex, family history of ACS and glucose levels. ROC curve analyzes revealed that exosomal Cyr61 expression could be used to differentiate patients with UAP, AMI and ACS from healthy individuals. Furthermore, exosomal Cyr61 levels were independently correlated with the existence of ACS. In vitro, Cyr61 expression was demonstrated to be significantly increased in VSMCs after ox-LDL exposure in a concentration- and time-dependent manner. Functionally, the elevated cell viability and migration of VSMCs induced by ox-LDL were partially but significantly inhibited by Cyr61 knockdown. By contrast, knocking down Cyr61 expression significantly elevated the apoptosis rate of VSMCs compared with that in the ox-LDL-treated group. In conclusion, data from the present study suggest that Cyr61 serve a regulatory role in vascular remodeling in vitro, where exosomal Cyr61 levels may represent a promising biomarker for ACS diagnosis.

ABSTRACTThe present study was designed to determine the role of PKD in vascular remodeling (VR) in Spontaneously hypertensive rats (SHRs). Increased SBP, VR and PKD activation were prominent in SHRs. The SBP has a positive correlation with the activation of PKD in SHRs. The ratio of media to lumen (MT/LD), volume fraction of collagen (VFC), hydroxyproline, IL-6, TNF-α and nitrotyrosine content were significantly related to the activated PKD. It may be concluded that PKD plays a central role in VR, and the mechanism may be related to its regulation of hypertrophy, fibrosis, inflammation and oxidative stress.

Background: Hypertension (HTN) and chronic kidney disease (CKD) are among the most prevalent global health conditions that cause millions of deaths per year. They are associated with vascular damage characterized by vascular remodeling, stiffening and endothelial dysfunction. miRNAs (miRs) are a class of small non-coding RNA that regulate gene expression by binding to their target messenger RNAs (mRNAs), thereby leading to mRNA degradation or translational repression. Their implication in vascular injury remains unclear. We aimed to identify differentially expressed (DE) miRs in small arteries of HTN and CKD human subjects to gather insight into pathophysiological molecular mechanisms in these conditions. Methods and Results: Normotensive, HTN [systolic blood pressure (BP) &gt;135 mmHg or diastolic BP of 85-115 mmHg with BpTRU] and CKD subjects (eGFR&lt;60mL/min/m 2 ) (n=15-16) were studied. Small arteries were isolated from subcutaneous gluteal biopsies and RNA extracted for small and total RNA sequencing using Illumina HiSeq-2500. EdgeR identified DE miRs ( P &lt;0.05) uniquely associated with HTN (3 up and 6 down) or CKD (42 up and 39 down) or with both groups (2 down). Reverse transcription-quantitative PCR (RT-qPCR) was used to confirm miRNA differential expression. Correlation between RNA sequencing and RT-qPCR data was demonstrated for 3 miRs from 14 tested: mir-146a-5p (r=0.56, P &lt;10 -4 ), miR-338-3p (r=0.91, P &lt;10 -16 ) and mir-374a-3p (r=0.57, P &lt;10 -4 ). The best correlated DE miRNA, miR-338-3p was down-regulated by 76% ( P &lt;10 -4 ) and uniquely associated with CKD. TargetScan predicted that the up-regulated mRNA encoding glutathione peroxidase 3 ( GPX3 , 1.57 fold, P &lt;0.001) to be a miR-338-3p target. Both miR-338-3p and GPX3 were found to be highly expressed in human aortic endothelial cells (HAECs) by RT-qPCR. Target prediction was validated by showing using RT-qPCR that GPX3 expression was up-regulated 1.73±0.28 fold in HAECs transfected with anti-miR-338-3p ( P &lt;0.05). Conclusion: miR-338-3p down-regulation was found in small arteries, uniquely associated with CKD. GPX3 , which may be important for preservation of endothelial function, is a miR-338-3p potential target. miR-338-3p and associated target may play a role in vascular remodeling in CKD.

Matrix metalloproteinases and their inhibitors in vascular remodeling and vascular disease

Abnormal proliferation of vascular smooth muscle cells (VSMCs) occurs in hypertension, atherosclerosis and restenosis after angioplasty, leading to pathophysiological vascular remodeling. As an important growth arrest gene, p21 plays critical roles in vascular remodeling. Regulation of p21 expression by retinoic acid receptor (RAR) and its ligand has important implications for control of pathological vascular remodeling. Nevertheless, the mechanism of RAR-mediated p21 expression in VSMCs remains poorly understood. Here, we show that, under basal conditions, RARα forms a complex with histone deacetylase 2 (HDAC2) and Krüppel-like factor 5 (Klf5) at the p21 promoter to inhibit its expression. Upon RARα agonist stimulation, HDAC2 is phosphorylated by CK2α. Phosphorylation of HDAC2, on the one hand, promotes its dissociation from RARα, thus allowing the liganded-RARα to interact with co-activators; on the other hand, it increases its interaction with Klf5, thus leading to deacetylation of Klf5. Deacetylation of Klf5 facilitates its dissociation from the p21 promoter, relieving its repressive effect on the p21 promoter. Interference with HDAC2 phosphorylation by either CK2α knockdown or the use of phosphorylation-deficient mutant of HDAC2 prevents the dissociation of Klf5 from the p21 promoter and impairs RAR agonist-induced p21 activation. Our results reveal a novel mechanism involving a phosphorylation-deacetylation cascade that functions to remove the basal repression complex from the p21 promoter upon RAR agonist treatment, allowing for optimum agonist-induced p21 expression.

Pericyte Requirement for Anti-Leak Action of Angiopoietin-1 and Vascular Remodeling in Sustained Inflammation

A proliferative and apoptosis-resistant phenotype in pulmonary arterial smooth muscle cells (PASMCs) is key to pathologic vascular remodeling in pulmonary hypertension (PH). Expression of indoleamine-2,3-dioxygenase (IDO) by vascular endothelium is a newly identified vasomotor-regulatory mechanism also involved in molecular signaling cascades governing vascular smooth muscle cell (vSMC) plasticity. To investigate the therapeutic potential of enhanced endothelial IDO in development of PH and its associated vascular remodeling. We used loss and gain of function in vivo studies to establish the role and determine the therapeutic effect of endothelial IDO in hypoxia-induced PH in mice and monocrotaline-induced PH in rats. We also studied PASMC phenotype in an IDO-high in vivo and in vitro tissue microenvironment. The endothelium was the primary site for endogenous IDO production within mouse lung, and the mice lacking this gene had exaggerated hypoxia-induced PH. Conversely, augmented pulmonary endothelial IDO expression, through a human IDO-encoding Sleeping Beauty (SB)-based nonviral gene-integrating approach, halted and attenuated the development of PH, right ventricular hypertrophy, and vascular remodeling in both preclinical models of PH. IDO derived from endothelial cells promoted apoptosis in PH-PASMCs through depolarization of mitochondrial transmembrane potential and down-regulated PH-PASMC proliferative/synthetic capacity through enhanced binding of myocardin to CArG box DNA sequences present within the promoters of vSMC differentiation-specific genes. Enhanced endothelial IDO ameliorates PH and its associated vascular structural remodeling through paracrine phenotypic modulation of PH-PASMCs toward a proapoptotic and less proliferative/synthetic state.

Atherosclerosis is a major cause for the death of human beings, and it takes place in large- and middle-sized arteries. The pathogenesis of the disease has been widely investigated, and new findings on vascular stem/progenitor cells could have an impact on vascular regeneration. Recent Advances: Recent studies have shown that abundant stem/progenitor cells present in the vessel wall are mainly responsible for cell accumulation in the intima during vascular remodeling. It has been demonstrated that the mobilization and recruitment of tissue-resident stem/progenitor cells give rise to endothelial and smooth muscle cells (SMCs) that participate in vascular repair and remodeling such as neointimal hyperplasia and arteriosclerosis. Interestingly, cell lineage tracing studies indicate that a large proportion of SMCs in neointimal lesions is derived from adventitial stem/progenitor cells. The influence of stem/progenitor cell behavior on the development of atherosclerosis is crucial. An understanding of the regulatory mechanisms that control stem/progenitor cell migration and differentiation is essential for stem/progenitor cell therapy for vascular diseases and regenerative medicine. Identification of the detailed process driving the migration and differentiation of vascular stem/progenitor cells during the development of atherosclerosis, discovery of the environmental cues, and signaling pathways that control cell fate within the vasculature will facilitate the development of new preventive and therapeutic strategies to combat atherosclerosis. Antioxid. Redox Signal. 00, 000-000.