Vascular Remodeling Process Research Articles

Tumor necrosis factor-related apoptosis-inducing ligand regulate the accumulation of extracelluar matrix in pulmonary artery by activating the phosphorylation of Smad2/3.

Introduction: Previous studies have found that tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) was involved in the progression of pulmonary hypertension (PH), and TRAIL knocking (KO) has an inhibitory effect on PH, but its mechanism is not completely clear. Methods: The effects of TRAIL on the accumulation of extracelluar matrix (ECM), which is one of the most important processes of vascular remodeling, were observed in mice and isolated pulmonary artery smooth muscle cells (PASMCs). In vivo, mice were divided into four groups: Control group (n = 5), hypoxia-induced PH mice group (n = 8), anti-TRAIL antibody (TRAIL-Ab) treatment group (n = 8) and IgG antibody (IgG) group (n = 8). The effects of TRAIL-Ab on ECM expression in hypoxic induced PH were researched; in vivo, PASMCs were divided into three groups: Control group, hypoxia-induced group, TRAIL-Ab group. Expressions of p-Smad2/3 and p-Smad1/5/8 were compared among the three groups. Results: Hypoxia-induced PH mice had significant increases in right ventricle systolic pressure (RVSP) (P < 0.001), right ventricular hypertrophy (RVH) (P = 0.007), vascular stenosis (P < 0.001) compared with controls. Mice with anti-TRAIL antibody had lower levels in RVSP (P < 0.001), RVH (P < 0.001), vascular stenosis (P < 0.001) than PH mice. Besides, the TRAIL-Ab significantly inhibited the phosphorylation of Smad2/3 compared with hypoxia-induced group. Conclusion: TRAIL regulates the accumulation of ECM in pulmonary artery by activating pSmad2/3.

Chronic dissection aneurysm of the aorta: clinical and pathologic study

An analysis of the clinical case of chronic dissection aortic aneurysm (DAA) in a 47-year-old man was conducted. Diagnosis of chronic DAA type A for Stanford is established at CT angiography of the thoracic and abdominal aorta with proven contrast enhancement. The operation of supracoronary prosthetics of the ascending department of the aorta was performed with full root method. During surgery, a chronic RAA of the ascending aorta with a diameter of about 5.5 cm was detected. The aorta was enlarged in the area of the root, whereas at the point of transition into the arc was of ordinary diameter. The aortic valve was three-lobed with signs of slight dilation of the fibrous ring. The ascending aorta was circularly mobilized and excised 1.0 cm above the coronary artery cells. The organized thrombus from the false lumen from the lateral wall of the ascending aorta (from the side of the superior vena cava) was removed. A proximal anastomosis was applied between the vascular prosthesis and the ascending aorta with a telescopic double continuous suture. Subsequently, a distal ascending aorta 1.5 cm below the shoulder-main trunk was dissected. Therefore, a distal anastomosis was applied between the distal ascending aorta and the vascular prosthesis with a telescopic double continuous suture. The postoperative period was satisfactory. On the 8th day of the postoperative period the patient was in a satisfactory condition discharged home.&#x0D; Pathomorphological examination of the operating material revealed segmental hyperplasia of the media with focal mucoid swelling, multiple cystic cavities, diffuse-focal fibrosis. The cavity of the dissection looked like a “two-stemmed” (two-channel) lumen, located on the border of adventitia and media, was filled with blood clots with the spread of hemorrhagic infiltration into the peri-adventitious fat. The new pseudocanal contained blood clots and fresh blood clots, and the old one was completely covered with endothelium with neointima formation. The aortic architecture is completely broken, the division into layers was absent. In the section of the irregular canal, the formation of a two-layer structure was observed – neointima, which differed from the intima of the true lumen of the aorta by the number of smooth muscle cells and heterogeneous accumulation of components of the extracellular matrix. In the middle layers, connective tissue remodeling of the aorta was detected in the form of chaotically located immature mesenchymal cells in combination with muscular and connective tissue hyperplasia, complete loss of elastic fibers, massive sclerosis of adventitia, and periadventitial layer. In sections of the aorta, out of stratification, its structure was preserved, there are signs of cystic medial necrosis.&#x0D; The revealed pathomorphological changes in the described case point to the processes of vascular remodeling, which explain the relative stability of DAA.

P6008Inhibin Beta-A is a novel gene involved in the development of pulmonary arterial hypertension through inhibiting BMPRII-signaling in endothelial cells

Abstract Introduction Pulmonary Arterial Hypertension (PAH) is marked by vascular remodeling process that eventually causes pressure increase. Endothelial cells (EC) dysfunction is known to be a major cause for pulmonary vascular remodeling; however, the molecular mechanism remains to be elucidated. Purpose This study aims to identify novel genes and mechanisms involved in PAH development. Methods We performed DNA microarray analysis using RNA samples isolated from human ECs of various vascular beds (including lung microvessels) and organs (including lung). We subsequently searched for genes highly and specifically expressed in lung microvessels since these genes are likely involved in pulmonary circulation homeostasis maintenance. Once found, we confirmed its expressional changes during hypoxia in ECs and lung tissues. We next analyzed its role in EC functions using human pulmonary artery ECs (hPAECs) by in vitro angiogenesis assay, using both candidate gene overexpression via retrovirus transfection and treatment with its active form using appropriate recombinant protein. To explore the role of candidate gene in PAH development in vivo, we generated EC-specific knockout mice and transgenic mice in which the candidate gene is genetically deleted and activated in ECs, respectively. PAH was induced by chronic hypoxia exposure (10% O2- for 3 weeks). Lastly, to explore the underlying mechanisms, we analyzed expressional alterations in possible signaling pathways in ECs that could relate with the effect of the candidate gene. Results From microarray analysis, we identified inhibin Beta-A (INHBA) as a candidate gene that was highly and specifically expressed in human lung microvascular ECs. INHBA homo-dimerization is known to produce activin A (ActA), a TGF-beta superfamily member. Hypoxia exposure caused significant decrease of INHBA mRNA expression in ECs and mouse lung tissues. Both INHBA overexpression and ActA-treatment in hPAECs caused dramatic reduction of their angiogenic capacities (reduced migration and tube formation capability with increased apoptosis). In vivo, EC-specific INHBA overexpressing mice (VEcad-INHBA-TG) showed exacerbated hypoxia-induced PAH, assessed by higher right ventricular systolic pressure (RVSP) and more severely remodeled pulmonary arteries. By contrast, EC-specific INHBA knockout mice (INHBA-floxed/VEcad-Cre-TG) showed significant amelioration of PAH, shown by reduced RVSP and vascular remodeling. Furthermore, we found that INHBA overexpression and ActA-treatment induced a marked reduction of BMPRII, known to play pivotal roles in PAH, in hPAECs by accelerating its lysosomal degradation. Conclusion We identified a novel gene that is crucially involved in PAH development. INHBA and/or ActA negatively regulates EC functions potentially through its BMPRII-altering capability. Gain- and loss-of-function studies in mice revealed that INHBA pathways are promising therapeutic targets for the treatment of PAH.

P4143IL-6 production in the vascular adventitia and adventitia-media-crosstalk in neointima formation

Abstract Background The aim of this study was to analyze the impact of the adventitial layer on vascular remodeling processes and to define the underlying cellular mechanisms. Methods and results Morphometric analysis of human coronary arteries and of murine femoral arteries at several times following vascular intervention revealed a significant correlation of neointimal and adventitial thickening (R2=0.6845, P&lt;0.001 for human samples; R2=0.6845, P&lt;0.001 for human samples. Immunohistochemical staining for the proliferation marker Ki-67 was performed 7, 14, and 21 days following injury of the murine femoral artery. Formation of a neointimal lesion at 21 days was preceded by high adventitial proliferation rates at 7 and 14 days (85.00±6.041 Ki67+adventitial cells vs. 5.118±0.633 Ki-67+neointimal cells at 7d, P&lt;0.0014; 28.80±5.240 Ki-67+adv. cells vs. 19.40±2.468 Ki-67+neoint. cells at 14d, P&lt;0.006, n=17). Complete removal of the adventitial layer prevented neointima formation, attributing pivotal importance to the adventitial layer (luminal stenosis: 71.73±3.77% vs. 7.44±1.71%, n=5, P&lt;0.0001). Re-transplantation of the aortic adventitia of ubiquitously GFP expressing C57BL/6-Tg (CAG-EGFP)1Osb/Jmice around the medial vascular layer of the femoral artery where the native adventitia has been removed completely restored neointima formation. Importantly, only very view GFP+cells were present in the neointimal layer, indicating that a direct contribution of adventitial cells to the neointimal lesion represents an extremely rare event. To investigate a potential paracrine effect of the activated adventitial layer, we explanted adventitial transplants 14 days following injury and transplantation and incubated the respective samples in serum-free media for 24 hours. BrdU incorporation assays and scratch wound assays revealed significantly increased proliferation and migration rates of human coronary artery SMCs in response to the supernatant of adventitial transplants compared to the supernatant of control samples. Further secretome analyses of the same adventitial supernatants identified predominantly interleukin (IL)-6 to trigger SMC proliferation and migration. Accordingly, serum-free media incubated with adventitial grafts of IL-6−/− mice prevented SMC proliferation and migration. Transplantation of the adventitia of IL-6−/− mice into C57BL/6J wild type mice was not sufficient to trigger neointima formation. Plain old balloon angioplasty, bare metal stent implantation, or drug-eluting stent implantation in swine coronary arteries and analysis for Ki-67+ cell counts supported the hypothesis in the large animal model and a more clinical setting. Conclusion Acute vascular injury is followed by an expansion of cytokine-producing adventitial cells, whose paracrine function and especially whose release of IL-6 is essential for the subsequent induction of the proliferation and migration of local SMC and thus for neointima formation.

P4146The six-transmembrane protein Stamp2 protects from hypoxia-induced pulmonary vascular remodeling and pulmonary hypertension via actions in mononuclear cells and CXCL12

Abstract Background The six-transmembrane protein of prostate (Stamp2) is a potent anti-inflammatory player in adipocytes and also in macrophages. Stamp2's actions in these cells protects from diet-induced diabetes and from atherosclerosis mice. As chronic inflammation is a hallmark of pulmonary arterial hypertension (PH), we sought to investigate the role of Stamp2 in PH. Methods and results Morphometric analyses of small pulmonary arteries after 3 weeks of chronic hypoxia (10% O2) showed aggravated pulmonary vascular remodeling in Stamp2−/− mice as compared to WT, demonstrated by a significantly reduced number of non-muscularized vessels and higher extent of fully-muscularized vessels. Consequently, right ventricular systolic pressure (RVSP, Millar catheter via right jugular vein) was significantly higher in Stamp2−/− mice (33.4±0.7 mmHg vs. 30.3±1.4, p&lt;0.05). As endothelial (EC) and smooth muscle cells (PASMC) are critical for remodeling processes in PH, the role of Stamp2 in these cells was explored. However, siRNA-mediated knock-down of Stamp2 in human microvascular EC had no effect on apoptotic susceptibility (CellDeath Detection ELISA), or release of IL-6 (qPCR). Furthermore, Stamp2-deficiency in isolated primary PASMC had no effect on proliferation (BrdU incorporation) and chemotaxis (modified Boyden chamber). As Stamp2 deficiency promotes higher expression of inflammatory cytokines (IL6, IL1b, MCP1, TNFa, CXCL12, qPCR) and increased numbers of CD68-positive cells in the lung, actions of Stamp2 in macrophages are potentially driving vascular remodeling in PH. To test this hypothesis, PASMC proliferation and chemotaxis were assessed in response to treatment with supernatants from primary thioglycolate-elicited peritoneal Stamp2−/− or WT-macrophages. These experiments revealed that supernatants from Stamp2−/− macrophages induced PASMC proliferation and chemotaxis significantly stronger, thus providing a link between inflammatory actions in Stamp2 deficiency and vascular remodeling. To gain further insights, a cytokine array was performed with supernatants from Stamp2−/− and WT-macrophages, revealing CXCL12 as the most relevant candidate. Experiments with neutralizing antibodies confirmed the role of CXCL12 in driving Stamp2's actions on vascular remodelling processes in PASMC. Importantly, Stamp2 expression (qPCR, western blot analyses) was significantly lower in the lung of humans with idiopathic PAH (IPAH), as well as in experimental PH in rats (monocrotalin, sugen/hypoxia) and in mice (hypoxia). Conclusions Stamp2 deficiency aggravates hypoxia-induced pulmonary vascular remodeling and pulmonary hypertension in mice. On the cellular level, actions of Stamp2 in macrophages drive vascular remodelling processes in smooth muscle cells via secreted factors such as CXCL12. The finding of decreased expression of Stamp2 in human and various experimental forms of PH points towards a general protective role of Stamp2.

261Decisive role of microRNA-494 in smooth muscle cell proliferation and vascular remodeling

Abstract Background Proliferation of vascular smooth muscle cells triggered by cytokines and growth factors is a main driver in the development of vascular proliferative diseases such as atherosclerosis and in-stent-restenosis after angioplasty. MicroRNAs (miR) are small noncoding RNAs that can inhibit the expression of multiple genes simultaneously. However, the contribution of microRNAs to the differential gene regulation that triggers vascular remodeling processes is not well understood. Methods and results Neointima formation was induced by a wire-mediated injury of the femoral artery in C57BL/6 mice. Microarray analysis of the developing neointimal lesion showed a strong reduction of miR-494 (0.411±0.04; p&lt;0.05) at 7 days after injury. In order to investigate the expression levels of miRs in vascular cells, human coronary artery smooth muscle cells (SMC), human coronary artery endothelial cells and human monocytes were analyzed via microarray analysis. Intriguingly, miR-494 was found to be predominantly expressed in SMC via microarray and qPCR analysis. The regulation of miR-494 expression was further analyzed after stimulation of SMC with 10%FCS. Following this mitogenic stimulation, mir-494 expression dropped robustly and significantly in a time-dependent manner at 6, 9, and 24 hours. To investigate the functional impact of miR-494 on SMC proliferation, miR-494 was overexpressed using miR-494-mimics (20μM). Overexpression of miR-494 significantly reduced the FCS-induced proliferation of SMC as assessed by BrdU-incorporation. In silico analyses of potential target genes for miR-494 identified ROCK1 and Survivin, both important molecules in the mitogenic response of SMC to cytokines and growth factors, as potential targets of miR-494. Indeed, ROCK1 and Survivin were found down-regulated on the mRNA and protein level after transfection of SMC with miR-494 mimics and both mulecules could be identified as direct targets using luciferase reporter assays. Following the specific inhibition of miR-494 by local application (in a perivascular thermos-responsive, self-degrading pluronic gel) of an in vivo stabilized Pre-miR-494 after wire-induced injury of the mouse femoral artery, SMC proliferation was significantly reduced, as assessed by Ki67 immunofluorescence (26.3% vs 11.2%; p&lt;0.05). Consistently, local application of Pre-miR-494 significantly reduced neointima formation (neointima/media ratio 2.31 in control vs 1.01 in treated animals; p&lt;0.01). Conclusion Our results show that mir-494 is strongly down-regulated in proliferating SMC in vitro as well as during neointimal lesion formation in vivo. Moreover, overexpression/ reconstitution of miR-494 levels effectively prevented SMC proliferation and neointima formation, indicating an important functional role of miR-494 in these processes. Hence, miR-494 may represent an attractive SMC-specific target for future therapeutic interventions for the treatment of vascular proliferative diseases.

Modulating EGFR-MTORC1-autophagy as a potential therapy for persistent fetal vasculature (PFV) disease

ABSTRACT Persistent fetal vasculature (PFV) is a human disease that results from failure of the fetal vasculature to regress normally. The regulatory mechanisms responsible for fetal vascular regression remain obscure, as does the underlying cause of regression failure. However, there are a few animal models that mimic the clinical manifestations of human PFV, which can be used to study different aspects of the disease. One such model is the Nuc1 rat model that arose from a spontaneous mutation in the Cryba1 (crystallin, beta 1) gene and exhibits complete failure of the hyaloid vasculature to regress. Our studies with the Nuc1 rat indicate that macroautophagy/autophagy, a process in eukaryotic cells for degrading dysfunctional components to ensure cellular homeostasis, is severely impaired in Nuc1 ocular astrocytes. Further, we show that CRYBA1 interacts with EGFR (epidermal growth factor receptor) and that loss of this interaction in Nuc1 astrocytes increases EGFR levels. Moreover, our data also show a reduction in EGFR degradation in Nuc1 astrocytes compared to control cells that leads to over-activation of the mechanistic target of rapamycin kinase complex 1 (MTORC1) pathway. The impaired EGFR-MTORC1-autophagy signaling in Nuc1 astrocytes triggers abnormal proliferation and migration. The abnormally migrating astrocytes ensheath the hyaloid artery, contributing to the pathogenesis of PFV in Nuc1, by adversely affecting the vascular remodeling processes essential to regression of the fetal vasculature. Herein, we demonstrate in vivo that gefitinib (EGFR inhibitor) can rescue the PFV phenotype in Nuc1 and may serve as a novel therapy for PFV disease by modulating the EGFR-MTORC1-autophagy pathway. Abbreviations ACTB: actin, beta; CCND3: cyclin 3; CDK6: cyclin-dependent kinase 6; CHQ: chloroquine; COL4A1: collagen, type IV, alpha 1; CRYBA1: crystallin, beta A1; DAPI: 4ʹ6-diamino-2-phenylindole; EGFR: epidermal growth factor receptor; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GFAP: glial fibrillary growth factor; KDR: kinase insert domain protein receptor; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; MKI67: antigen identified by monoclonal antibody Ki 67; MTORC1: mechanistic target of rapamycin kinase complex 1; PARP: poly (ADP-ribose) polymerase family; PCNA: proliferating cell nuclear antigen; PFV: persistent fetal vasculature; PHPV: persistent hyperplastic primary vitreous; RPE: retinal pigmented epithelium; RPS6: ribosomal protein S6; RPS6KB1: ribosomal protein S6 kinase, polypeptide 1; SQSTM1/p62: sequestome 1; TUBB: tubulin, beta; VCL: vinculin; VEGFA: vascular endothelial growth factor A; WT: wild type.

Establishment and evaluation of hypoxia-induced mouse model of bronchopulmonary dysplasia associated with pulmonary hypertension

Objective To establish an animal model of hypoxia-induced bronchopulmonary dysplasia asso-ciated with pulmonary hypertension (BPD-PH). Methods C57BL/6 male and female specific pathogen free mice mated and female mice with their offspring mice were randomly divided into normoxic group and hypoxia group by way of numerical method.Normoxic group was placed in the indoor environment directly.Hypoxia group was placed in 120 mL/L oxygen concentration environment within 12 hours after birth.Body weight gain and mortality of the neonatal mice were recorded.The mice lungs and hearts were harvested on day 14 for immunofluorescence staining and HE staining, and Western blot was used to observe the morphological changes and vascular endothelial growth factor (VEGF) protein level. Results The mortality rates of normoxic group and hypoxic group were 11.8% and 47.3%, respectively.Compared with the normoxic group, body weight of hypoxia group increased slowly, as the final body weight of 2 groups were (12.40±2.33) g and (5.50±0.32) g, respectively, and the difference was significant (χ2=13.38, t=20.50, all P<0.01). Hypoxia group showed higher right ventricular hypertrophy index [(96.00±0.15)% vs.(40.40±4.00)%, t=41.67, P<0.01], fusion of alveolar, diminished microvasculature, thickening alveolar septal, decreased alveolar radiation coefficient (RAC)(19.73±2.33 vs.10.90±1.85) and increased mean linear intercept(MLI) (33.2±4.33 vs.58.70±7.27) with statistical significance, respectively (t=16.27, 9.53, all P<0.01). In the lung HE staining, pulmonary arterial thickening, pulmonary smooth muscle cell proliferation and intimal roughness in immunofluorescence, significantly decreased expression of VEGF protein level by Western blot were observed in hypoxic group (0.41±0.04 vs.1.19±0.08, t=15.10, P<0.01). Conclusions Hypoxia-induced mouse BPD-PH model was consistent with the pathophysiological process of pulmonary vascular remodeling of pulmonary hypertension, which increase pulmonary vascular resistance eventually leading to right ventricular hypertrophy. Key words: Bronchopulmonary dysplasia; Hypoxia; Pulmonary hypertension; Mice

C-Kit deficiency impairs nitric oxide signaling in smooth muscle cells

IntroductionReceptor tyrosine kinases have been implicated in various vascular remodeling processes and cardiovascular disease. However, their role in the regulation of vascular tone is poorly understood. Herein, we evaluate the contribution of c-Kit signaling to vasoactive responses. MethodsThe vascular reactivity of mesenteric arteries was assessed under isobaric conditions in c-Kit deficient (KitW/W−v) and littermate control mice (Kit+/+) using pressure myography. Protein levels of soluble guanylyl cyclase beta 1 (sGCβ1) were quantified by Western blot. Mean arterial pressure was measured after high salt (8% NaCl) diet treatment using the tail-cuff method. ResultsSmooth muscle cells (SMCs) from c-Kit deficient mice showed a 5-fold downregulation of sGCβ1 compared to controls. Endothelium-dependent relaxation of mesenteric arteries demonstrated a predominance of prostanoid vs. nitric oxide (NO) signaling in both animal groups. The dependence on prostanoid-induced dilation was higher in c-Kit mutant mice than in controls, as indicated by a significant impairment in vasorelaxation with indomethacin with respect to the latter. Endothelium-independent relaxation showed significant dysfunction of NO signaling in c-Kit deficient SMCs compared to controls. Mesenteric artery dilation was rescued by addition of a cGMP analog, but not with a NO donor, indicating a deficiency in cGMP production in c-Kit deficient SMCs. Finally, c-Kit deficient mice developed higher blood pressure on an 8% NaCl diet compared to their control littermates. Conclusionc-Kit deficiency inhibits NO signaling in SMCs. The existence of this c-Kit/sGC signaling axis may be relevant for vascular reactivity and remodeling.

Abstract 794: Endothelial Specific Ablation of Estrogen Receptor Alpha Rapid Signaling Revealed Exacerbated Vascular Remodeling Response

Background: Estrogen exerts complex physiological effects via its rapid (non-genomic) and genomic actions. In particular, rapid signaling of estrogen receptor alpha (ERα) has been implicated in the vasculo-protective effects, in which both endothelial and smooth muscle cells might be involved. However, no prior studies have determined the role of ERα rapid signaling in the endothelium. This study aims to clarify the impact of ERα rapid signaling in the vasculo-protection, using a novel mouse model lacking endotheilal-specific ERα rapid signaling. Methods and Results: In immunoblotting, p85α and pGSK3β, non-genomic pathway downstream signals, were reduced in ERα mutants RR259/260AA with estradiol (E2) stimulation at concentrations of 10 -11 and 10 -10 M . ERE-luciferase assay demonstrated E2 induced genomic pathway activity was preserved even in ERα mutants RR259/260AA. We generated a mouse model in which rapid signaling of ERα was ablated in the endothelium by crossing Tie2-Cre transgenic mice with floxed ERα mutants (RR 259/260 AA) in which p85α and ERα interaction was disrupted. In endothelial cells isolated from ERα KI/KI Tie2 cre/+ animals, E2 at a concentration of 10 -8 M failed to induce phosphorylation of Akt, confirming the absence of ERα rapid signaling. Baseline characteristics at 8 to 12 weeks of age were undistinguishable between the genotypes, including body weight (KI:19.2±0.366 g vs WT: 18.9±0.356 g ) systolic blood pressure (KI:106.1±3.336 vs WT: 103.5±2.133 mmHg) and echocardiographic fractional shortening (KI:54.4±0.751 vs WT: 55.19±0.947 %). We then assessed how vascular remodeling process was impacted in a carotid artery wire injury model. Histological analyses with Elastica van Gieson staining two weeks after induction of injury revealed that wall thickness and area of medial layer composed mainly of smooth muscle cells were significantly increased in ERα KI/KI Tie2 cre/+ mouse, as compared to wild types (KI:44.85±1.948 μm vs WT: 29.32±1.753 μm , KI:42,047±1,560 μm 2 vs WT: 32,815±1,211 μm 2 , respectively ). Conclusions: Our results demonstrate that the rapid signaling of ERα in the endothelium critically regulates vascular smooth muscle cell growth after vascular injury, suggesting the essential role to vascular remodeling.

Hypoxia inducible factor 1\u03b1 in vascular smooth muscle cells promotes angiotensin II-induced vascular remodeling via activation of CCL7-mediated macrophage recruitment

The process of vascular remodeling is associated with increased hypoxia. However, the contribution of hypoxia-inducible factor 1α (HIF1α), the key transcription factor mediating cellular hypoxic responses, to vascular remodeling is established, but not completely understood. In the angiotensin II (Ang II)-induced vascular remodeling model, HIF1α was increased and activated in vascular smooth muscle cells (VSMCs). Selective genetic disruption of Hif1a in VSMCs markedly ameliorated Ang II-induced vascular remodeling, as revealed by decreased blood pressure, aortic thickness, collagen deposition, inflammation, and aortic stiffness. VSMC Hif1a deficiency also specifically suppressed Ang II-induced infiltration of CD45+CD11b+F4/80+CD206− M1 macrophages into the vessel. Mechanistically, HIF1α deficiency in VSMCs dramatically suppressed the expression of CCL7, a chemokine critical for macrophage recruitment. Bioinformatic analysis and chromatin immunoprecipitation assays revealed three functional hypoxia-response elements in the Ccl7 promoter, indicating that Ccl7 is a direct HIF1α target gene. Blocking CCL7 with antibody in vivo alleviated Ang II-induced hypertension and vascular remodeling, coincident with decreased macrophage infiltration. This study provides direct evidence that HIF1α activation in VSMCs exacerbates Ang II-induced macrophage infiltration and resultant vascular remodeling via its target gene Ccl7, and thus may serve as a potential therapeutic target for remodeling-related vascular disease.

Plasticity of vascular resident mesenchymal stromal cells during vascular remodeling

Vascular remodeling is a complex and dynamic pathological process engaging many different cell types that reside within the vasculature. Mesenchymal stromal/stem cells (MSCs) refer to a heterogeneous cell population with the plasticity to differentiate toward multiple mesodermal lineages. Various types of MSC have been identified within the vascular wall that actively contribute to the vascular remodeling process such as atherosclerosis. With the advances of genetic mouse models, recent findings demonstrated the crucial roles of MSCs in the progression of vascular diseases. This review aims to provide an overview on the current knowledge of the characteristics and behavior of vascular resident MSCs under quiescence and remodeling conditions, which may lead to the development of novel therapeutic approaches for cardiovascular diseases.

Role of matrix metalloproteinases in the pathogenesis of intracranial aneurysms.

Intracranial aneurysms (IAs) are a result of complex interactions between biochemical and mechanical forces and can lead to significant morbidity if they rupture and cause subarachnoid hemorrhage. This review explores the role of matrix metalloproteinases (MMPs) in the pathogenesis and progression of IAs. In addition to providing a review of the normal function of MMPs, it is intended to explore the interaction between inflammation and abnormal blood flow and the resultant pathological vascular remodeling processes seen in the development and rupture of IAs. Also reviewed is the potential for the use of MMPs as a diagnostic tool for assessment of aneurysm development and progression.

Oligonucleotide Microarrays Identified Potential Regulatory Genes Related to Early Outward Arterial Remodeling Induced by Tissue Plasminogen Activator.

Constrictive vascular remodeling limiting blood flow, as well as compensatory outward remodeling, has been observed in many cardiovascular diseases; however, the underlying mechanisms regulating the remodeling response of the vessels remain unclear. Plasminogen activators (PA) are involved in many of the processes of vascular remodeling. We have shown previously that increased levels of tissue-type PA (tPA) contributes to outward vascular remodeling. To elucidate the mechanisms involved in the induction of outward remodeling we characterized changes in the expression profiles of 8799 genes in injured rat carotid arteries 1 and 4 days after recombinant tPA treatment compared to vehicle. Periadventitial tPA significantly increased lumen size and vessel area, encompassed by the external elastic lamina, at both one and 4 days after treatment. Among 41 differentially expressed known genes 1 day after tPA application, five genes were involved in gene transcription, five genes were related to the regulation of vascular tone [for example, thromboxane A2 receptor (D32080) or non-selective-type endothelin receptor (S65355)], and eight genes were identified as participating in vascular innervation [for example, calpain (D14478) or neural cell adhesion molecule L1 (X59149)]. Four days after injury in tPA-treated arteries, four genes, regulating vascular tone, were differentially expressed. Thus, tPA promotes outward arterial remodeling after injury, at least in part, by regulating expression of genes in the vessel wall related to function of the nervous system and vascular tone.

Osteopontin lung gene expression is a marker of disease severity in pulmonary arterial hypertension.

Osteopontin (OPN) is a pleiotropic cytokine involved in the proliferation of pulmonary artery smooth muscle cells (PA-SMC). OPN is upregulated in the lungs of patients with pulmonary hypertension (PH) associated with pulmonary fibrosis, suggesting that the lung is a source of OPN. We hypothesized that OPN lung expression is elevated in Group I pulmonary arterial hypertension (PAH) and is correlated to haemodynamics. Microarray analysis (Affymetrix) was performed after RNA was extracted from explanted lungs in 15 patients with Group I PAH who underwent lung transplantation (LTx) and 11 normal controls. PA pressure levels were recorded intraoperatively, immediately before starting LTx. Serum OPN levels were measured in subjects with PAH, Group II PH and normal controls on the day of right heart catheterization. OPN was among the top five upregulated genes in PAH compared to normal controls, which was confirmed by reverse transcription polymerase chain reaction (RT-PCR). OPN expression was similar and equally elevated in different subtypes of PAH. A strong significant correlation was observed between mean pulmonary arterial pressure and OPN gene expression. Ingenuity pathway analysis showed the involvement of OPN in functions and networks relevant to angiogenesis, cell death and proliferation of PA-SMC. OPN serum levels did not differ in subjects with Group I PAH and Group II PH. In the lungs of patients with severe PAH, OPN is highly expressed and the level of expression is significantly correlated to disease severity. OPN may play an important role in the vascular remodelling process of PAH.

LIM Kinase Inhibition Diminishes Hypertension and Vasoconstriction‐Induced Inward Remodeling in Mouse and Human Resistance Arteries

Vascular remodeling is an early biomarker of cardiovascular disease (CVD) related to structural changes in resistance arteries (RA). Arteriolar inward remodeling, characterized by a reduced luminal diameter that is associated with changes in the homeostasis of actin polymerization/depolymerization pathways, is posited to precede the development of hypertension. We hypothesize that inward remodeling involves changes in the actin cytoskeleton that facilitate cell motility and the rearrangement of vascular smooth muscle cells (VSMCs) around the vascular lumen. Moreover, we predict that inhibition of actin polymerization with a Lim kinase (LIMK) inhibitor would block the inward remodeling process associated with prolonged vasoconstriction and hypertension. We have previously reported in ex vivo experiments in rats that long‐term exposure of RA to vasoconstrictors induces vascular inward remodeling via activation of the Rho pathway, which in part promotes actin polymerization. We now demonstrate that VSMCs increase the inward remodeling ratio of filamentous to globular actin (F/G‐actin) in response to GPCR‐activation by a norepinephrine (NE) and angiotensin‐II (AngII). GPCR‐mediated Rho activates (LIMK), which subsequently phosphorylates and inactivates the actin severing enzyme, cofilin, thus effecting an increase in F/G‐actin ratios. In isolated rat cremasteric RA we have demonstrated that LIMK inhibition blocks the inward remodeling induced by of GPCR agonists and that this is coincident with a reduction in cofilin phosphorylation. To study the arterial remodeling process in vivo, we imaged the outer ear of a transgenic mouse line that expresses smooth muscle actin‐GFP in VSMCs by multiphoton microscopy. We treated these mice with AngII (1000ng/kg/min) for 14 days to induce vasoconstriction and hypertension. We imaged the same resistance artery within each ear before (Day 0) and after (Day 14) AngII infusion to study the progression of remodeling over time. Starting at day 0, one ear was injected every four days with a LIMK inhibitor (LIMKi), while the other ear received vehicle (control). We observed a significant inward remodeling in vessels of the control ear compared to those of the ear treated with LIMKi (p&lt;0.05). We then assessed the effect of LIMKi in the inward remodeling process associated with ex vivo prolonged vasoconstriction in isolated human RA from omental tissues and studied by pressure myography under passive conditions before and after a 4h incubation in the presence of NE + AngII (control) or NE + AngII + LIMKi. Exposure to NE + AngII induced vascular inward remodeling that was completely ablated by presence of LIMKi and resulted in outward remodeling (p&lt;0.05). These data indicate that LIMK plays a necessary role in the vasoconstriction‐hypertension vascular remodeling process that makes it a potential target for the treatment of CVDs associated with resistance artery inward remodeling.Support or Funding InformationNational Institutes of Health grants: R01 HL137769 (JP), R01 HL088105 (LM‐L).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

9.4T Magnetic Resonance Imaging of the Mouse Circle of Willis Enables Serial Characterization of Flow-Induced Vascular Remodeling by Computational Fluid Dynamics.

The neurovasculature dynamically responds to changes in cerebral blood flow by vascular remodeling processes. Serial imaging studies in mouse models could help characterize pathologic and physiologic flow-induced remodeling of the Circle of Willis (CoW). We induced flow-driven pathologic cerebral vascular remodeling in the CoW of mice (n=3) by ligation of the left Common Carotid Artery (CCA), and the right external carotid and pterygopalatine arteries, increasing blood flow through the basilar and the right internal carotid arteries. One additional mouse was used as a wild-type control. Magnetic Resonance Imaging (MRI) at 9.4 Tesla (T) was used to serially image the mouse CoW over three months, and to obtain threedimensional images for use in Computational Fluid Dynamic (CFD) simulations. Terminal vascular corrosion casting and scanning electron microscope imaging were used to identify regions of macroscopic and microscopic arterial damage. We demonstrated the feasibility of detecting and serially measuring pathologic cerebral vascular changes in the mouse CoW, specifically in the anterior vasculature. These changes were characterized by bulging and increased vessel tortuosity on the anterior cerebral artery and aneurysm- like remodeling at the right olfactory artery origin. The resolution of the 9.4T system further allowed us to perform CFD simulations in the anterior CoW, which showed a correlation between elevated wall shear stress and pathological vascular changes. In the future, serial high-resolution MRI could be useful for characterizing the flow environments corresponding to other pathologic remodeling processes in the mouse CoW, such as aneurysm formation, subarachnoid hemorrhage, and ischemia.

Galectin-3 Mediates Endothelial-to-Mesenchymal Transition in Pulmonary Arterial Hypertension.

Galectin-3 (Gal-3) is highly expressed in fibrotic tissue related to diverse etiologies. endothelial-to-mesenchymal transition (EndoMT), A less well studied phenomenon serves as a critical process in pulmonary vascular remodeling associated with the development of pulmonary arterial hypertension (PAH). EndoMT is hypothesized to contribute to the over-proliferation of αSMA positive cells. We aim to investigate the potential role of Gal-3 in regulating EndoMT in PAH. We observed an upregulation in both Gal-3 and αSMA expression in the monocrotaline (MCT) and Hypoxia PAH model, accompanied with intimal thickening. For more profound vascular remodeling and endothelial layer lesion in former model, we employed Gal-3 knockdown and overexpression lentivirus methodology to the MCT rats to determine the mechanisms underlying abnormal endothelial cell transition in PAH. PAH was evaluated according to right ventricular systolic pressure, right heart hypertrophy and pulmonary artery remodeling. A reduction in Gal-3 was protective against the development of PAH, while Gal-3 upregulation aggravated pulmonary vascular occlusion. In addition, Gal-3 deficiency suppressed pulmonary vascular cell proliferation and macrophage infiltration. Finally, we revealed that in endothelial cells treated with tumor necrosis factor α and hypoxia (representing an in vitro model of PAH), inhibition of Gal-3 by siRNA was able to abolish the associated upregulation of αSMA. These observations suggesting Gal-3 serves as a critical mediator in PAH by regulating EndoMT. Inhibition of Gal-3 may represent a novel therapeutic target for PAH treatment.

Vascular Remodeling Process of Heparin-Conjugated Poly(ε-Caprolactone) Scaffold in a Rat Abdominal Aorta Replacement Model

In the field of vascular graft research, poly-ε-caprolactone (PCL) is used owing to its good mechanical strength and biocompatibility. In this study, PCL scaffold was prepared by electrospinning and surface modification with heparin via hexamethylenediamine. Then the scaffolds were implanted into the infrarenal abdominal aorta of Wistar rats and contrast-enhanced micro-ultrasound was used to monitor the patency of grafts after implantation. These grafts were extracted from the rats at 1, 3, and 6 months for histological analysis, immunofluorescence staining, and scanning electron microscopy observation. Although some grafts experienced aneurysmal change, results showed that all implanted grafts were patent during the course of 6 months and these grafts demonstrated well-organized neotissue with endothelium formation, smooth muscle regeneration, and extracellular matrix formation. Such findings confirm feasibility to create heparin-conjugated scaffolds of next-generation vascular grafts.

Glycoprotein M6B Interacts with TβRI to Activate TGF-β-Smad2/3 Signaling and Promote Smooth Muscle Cell Differentiation.

Smooth muscle cells (SMCs), which form the walls of blood vessels, play an important role in vascular development and the pathogenic process of vascular remodeling. However, the molecular mechanisms governing SMC differentiation remain poorly understood. Glycoprotein M6B (GPM6B) is a four‐transmembrane protein that belongs to the proteolipid protein family and is widely expressed in neurons, oligodendrocytes, and astrocytes. Previous studies have revealed that GPM6B plays a role in neuronal differentiation, myelination, and osteoblast differentiation. In the present study, we found that the GPM6B gene and protein expression levels were significantly upregulated during transforming growth factor‐β1 (TGF‐β1)‐induced SMC differentiation. The knockdown of GPM6B resulted in the downregulation of SMC‐specific marker expression and repressed the activation of Smad2/3 signaling. Moreover, GPM6B regulates SMC Differentiation by Controlling TGF‐β‐Smad2/3 Signaling. Furthermore, we demonstrated that similar to p‐Smad2/3, GPM6B was profoundly expressed and coexpressed with SMC differentiation markers in embryonic SMCs. Moreover, GPM6B can regulate the tightness between TβRI, TβRII, or Smad2/3 by directly binding to TβRI to activate Smad2/3 signaling during SMC differentiation, and activation of TGF‐β‐Smad2/3 signaling also facilitate the expression of GPM6B. Taken together, these findings demonstrate that GPM6B plays a crucial role in SMC differentiation and regulates SMC differentiation through the activation of TGF‐β‐Smad2/3 signaling via direct interactions with TβRI. This finding indicates that GPM6B is a potential target for deriving SMCs from stem cells in cardiovascular regenerative medicine. stem cells 2018 Stem Cells 2019;37:190–201