Aortic Fibroblasts Research Articles

Paradoxical Changes: EMMPRIN Tissue and Plasma Levels in Marfan Syndrome-Related Thoracic Aortic Aneurysms.

Background: Thoracic aortic aneurysms (TAAs) associated with Marfan syndrome (MFS) are unique in that extracellular matrix metalloproteinase inducer (EMMPRIN) levels do not behave the way they do in other cardiovascular pathologies. EMMPRIN is shed into the circulation through the secretion of extracellular vesicles. This has been demonstrated to be dependent upon the Membrane Type-1 MMP (MT1-MMP). We investigated this relationship in MFS TAA tissue and plasma to discern why unique profiles may exist. Methods: Protein targets were measured in aortic tissue and plasma from MFS patients with TAAs and were compared to healthy controls. The abundance and location of MT1-MMP was modified in aortic fibroblasts and secreted EMMPRIN was measured in conditioned culture media. Results: EMMPRIN levels were elevated in MFS TAA tissue but reduced in plasma, compared to the controls. Tissue EMMPRIN elevation did not induce MMP-3, MMP-8, or TIMP-1 expression, while MT1-MMP and TIMP-2 were elevated. MMP-2 and MMP-9 were reduced in TAA tissue but increased in plasma. In aortic fibroblasts, EMMPRIN secretion required the internalization of MT1-MMP. Conclusions: In MFS, impaired EMMPRIN secretion likely contributes to higher tissue levels, influenced by MT1-MMP cellular localization. Low EMMPRIN levels, in conjunction with other MMP analytes, distinguished MFS TAAs from controls, suggesting diagnostic potential.

Abstract P1047: The Extracellular Matrix Metalloproteinase Inducer And The Membrane Type 1 Matrix Metalloproteinase In Marfan Syndrome Related Thoracic Aortic Aneurysm

Objectives: Marfan Syndrome (MFS) is a connective tissue disorder commonly associated with Thoracic Aortic Aneurysms (TAAs). TAAs occur because of abnormal remodeling of the aortic extracellular matrix (ECM). The balance between matrix metalloproteinases (MMPs) and the Tissue Inhibitors of MMPs (TIMPs), regulates these processes. The extracellular MMP inducer (EMMPRIN), is a widely expressed glycoprotein capable of stimulating the production of various MMPs, suggesting a direct role in driving pathology. The Membrane Type-1 MMP (MT1-MMP) can cause proteolytic shedding of EMMPRIN. Therefore, we investigated this relationship in MFS TAA tissue and plasma and tested the hypothesis that a unique proteolytic profile may exist. Methods: Levels of EMMPRIN, MT1-MMP, MMP-2, MMP-9, MMP-3, MMP-8, TIMP-1, and TIMP-2 were measured in aortic tissue and plasma from MFS patients with TAA and compared to healthy controls. Following modification of the abundance and location of MT1-MMP in isolated primary aortic fibroblasts, secreted EMMPRIN was measured in conditioned culture media. Results: Compared to controls, EMMPRIN is elevated in MFS TAA tissue and reduced in the plasma. Elevation of EMMPRIN abundance in tissue does not induce MMP-3, MMP-8, or TIMP-1 expression. MT1-MMP and TIMP-2 are elevated in MFS TAA tissue. MMP-2 and MMP-9 are reduced in MFS TAA tissue while increased in the plasma. In aortic fibroblasts we found that MT1-MMP must be internalized from the plasma membrane for EMMPRIN secretion. Conclusions: Our findings indicate that the pathological consequences of MFS cause the shedding of EMMPRIN and its secretion from cells to be blocked, which leads to higher EMPRINN abundance in MFS TAA tissue. This process depends upon the cellular location of MT1-MMP. Taken together, these observations suggest that internalization of MT1-MMP from the plasma membrane is required for the cellular secretion of EMMPRIN from aortic fibroblasts.

The angiopoietin receptor Tie2 is atheroprotective in arterial endothelium

Leukocytes and resident cells in the arterial wall contribute to atherosclerosis, especially at sites of disturbed blood flow. Expression of endothelial Tie1 receptor tyrosine kinase is enhanced at these sites, and attenuation of its expression reduces atherosclerotic burden and decreases inflammation. However, Tie2 tyrosine kinase function in atherosclerosis is unknown. Here we provide genetic evidence from humans and from an atherosclerotic mouse model to show that TIE2 is associated with protection from coronary artery disease. We show that deletion of Tie2, or both Tie2 and Tie1, in the arterial endothelium promotes atherosclerosis by increasing Foxo1 nuclear localization, endothelial adhesion molecule expression and accumulation of immune cells. We also show that Tie2 is expressed in a subset of aortic fibroblasts, and its silencing in these cells increases expression of inflammation-related genes. Our findings indicate that unlike Tie1, the Tie2 receptor functions as the dominant endothelial angiopoietin receptor that protects from atherosclerosis.

Abstract P124: Bainiku-ekisu Attenuates Cardiac Remodeling In Angiotensin Ii-infused Mice

A fruit of the Prunus mume is a traditional and familiar food in Asian countries and is most often pickled or used to make wine and juice concentrate. Recently, Bainiku-ekisu, an infused juice concentrate of Prunus mume is attracting attention as a potential health promoting food. We previously reported that Bainiku-ekisu (BE) incubation attenuates the activation of growth-promoting signaling cascade induced by angiotensin II (Ang II) in cultured vascular smooth muscle cells (VSMC). However, whether BE has any effect on an animal model of cardiovascular disease remains unknown. Therefore, this study was designed to elucidate whether BE could attenuate cardiovascular pathology induced by Ang II infusion in mice, representing a model of human hypertension. 8- to 10-week-old male C57BL6 mice were randomly divided into four groups and infused with Ang II (1 μg/kg/min) for 2 weeks via osmotic mini-pump or sham-operated, and given 1% BE containing water (BEW) or normal water (control) for 2 weeks. Blood pressure was evaluated every other day by tail cuff measurement. Cardiac hypertrophy was assessed by heart-to-body weight ratio. After 2 weeks, mice were euthanized, and the aorta and heart collected. Extracted aortas and hearts were fixed and used for histological studies to evaluate vascular hypertrophy and perivascular fibrosis. Increase in heart-to-body weight ratios were unaltered by BEW. Aortic medial hypertrophy was observed in control mice (n=5) after 2 weeks of Ang II infusion, which was attenuated in BEW group (96 μm vs. 71 μm, respectively, p<0.01). Control mice with angiotensin II infusion showed cardiac perivascular fibrosis, which was attenuated in BEW group (fibrosis index score: 1.02 vs. 0.30, p<0.01). In addition, trend of hypertension attenuation by BEW was observed in Ang II-infused mice with BEW treatment. We also observed attenuation of DNA synthesis induced by AngII with BE incubation in cultured aortic fibroblasts. In conclusion, BEW prevented Ang II-induced cardiovascular remodeling such as vascular fibrosis. Potential cardiovascular health benefit to taking BE as a functional food should be further studied.

RETRACTED: Fruit‐juice Concentrate of Asian Plum Attenuates Vascular Remodeling Associated with Hypertension

A fruit of the Prunus mume is a traditional and familiar food in Asian countries and is most often pickled or used to make wine and juice concentrate. Recently, Bainiku‐ekisu, an infused juice concentrate of Prunus mume is attracting attention as a potential health promoting food. We previously reported that Bainiku‐ekisu (BE) incubation attenuates the activation of growth‐promoting signaling cascade induced by angiotensin II (Ang II) in cultured vascular smooth muscle cells (VSMC). However, whether BE has any effect on an animal model of cardiovascular disease remains unknown. Therefore, this study was designed to elucidate whether BE could attenuate cardiovascular pathology induced by Ang II infusion in mice, representing a model of human hypertension. 8‐ to 10‐week‐old male C57BL6 mice were randomly divided into four groups and infused with Ang II (1 µg/kg/min) for 2 weeks via osmotic mini‐pump or sham‐operated, and given 1% BE containing water (BEW) or normal water (control) for 2 weeks. Blood pressure was evaluated every other day by tail cuff measurement. Cardiac hypertrophy was assessed by heart‐to‐body weight ratio. After 2 weeks, mice were euthanized, and the aorta and heart collected. Extracted aortas and hearts were fixed and used for histological studies to evaluate vascular hypertrophy and perivascular fibrosis. Increase in heart‐to‐body weight ratios were unaltered by BEW. Aortic medial hypertrophy was observed in control mice (n=5) after 2 weeks of Ang II infusion, which was attenuated in BEW group (96 µm vs. 71 µm, respectively, p<0.01). Control mice with angiotensin II infusion showed cardiac perivascular fibrosis, which was attenuated in BEW group (fibrosis index score: 1.02 vs. 0.30, p<0.01). In addition, trend of hypertension attenuation by BEW was observed in Ang II‐infused mice with BEW treatment. We also observed attenuation of DNA synthesis induced by AngII with BE incubation in cultured aortic fibroblasts. In conclusion, BEW prevented Ang II‐induced cardiovascular remodeling such as vascular fibrosis. Potential cardiovascular health benefit to taking BE as a functional food should be further studied.

Validation of Specific and Reliable Genetic Tools to Identify, Label, and Target Cardiac Pericytes in Mice.

BackgroundIn the myocardium, pericytes are often confused with other interstitial cell types, such as fibroblasts. The lack of well‐characterized and specific tools for identification, lineage tracing, and conditional targeting of myocardial pericytes has hampered studies on their role in heart disease. In the current study, we characterize and validate specific and reliable strategies for labeling and targeting of cardiac pericytes.Methods and ResultsUsing the neuron‐glial antigen 2 (NG2)DsRed reporter line, we identified a large population of NG2+ periendothelial cells in mouse atria, ventricles, and valves. To examine possible overlap of NG2+ mural cells with fibroblasts, we generated NG2DsRed; platelet‐derived growth factor receptor (PDGFR) αEGFP pericyte/fibroblast dual reporter mice. Myocardial NG2+ pericytes and PDGFRα+ fibroblasts were identified as nonoverlapping cellular populations with distinct transcriptional signatures. PDGFRα+ fibroblasts expressed high levels of fibrillar collagens, matrix metalloproteinases, tissue inhibitor of metalloproteinases, and genes encoding matricellular proteins, whereas NG2+ pericytes expressed high levels of Pdgfrb, Adamts1, and Vtn. To validate the specificity of pericyte Cre drivers, we crossed these lines with PDGFRαEGFP fibroblast reporter mice. The constitutive NG2Cre driver did not specifically track mural cells, labeling many cardiomyocytes. However, the inducible NG2CreER driver specifically traced vascular mural cells in the ventricle and in the aorta, without significant labeling of PDGFRα+ fibroblasts. In contrast, the inducible PDGFRβCreER line labeled not only mural cells but also the majority of cardiac and aortic fibroblasts.ConclusionsFibroblasts and pericytes are topographically and transcriptomically distinct populations of cardiac interstitial cells. The inducible NG2CreER driver optimally targets cardiac pericytes; in contrast, the inducible PDGFRβCreER line lacks specificity.

The endothelial-enriched lncRNA LINC01235 regulates hypoxia response via HIF-3a

Abstract Background Cardiovascular diseases (CVDs) remain the leading cause of death worldwide. Hypoxia induces significant changes in cardiovascular control mechanisms potentially resulting in pathophysiology. Recently, an increasing number of long non-coding RNAs (lncRNAs) was reported to participate in the regulation of Hypoxia-inducible factors (HIF). Analysis of single-cell RNA-sequencing of human Abdominal Aortic Aneurysms pinpointed the endothelial-enriched lncRNA LINC01235. LINC01235 was previously correlated with tumour progression in gastric cancer and worse patient prognosis in breast cancer. Globally, the role of LINC01235 in the cardiovascular system remains unknown. Purpose The objective of this study is to unravel the function of LINC01235 in endothelial cells (ECs). Methods and results LINC01235 levels were elevated in human umbilical vein ECs (7.66 fold, p<0.05), human aortic ECs (16.84 fold, p<0.05) and human dermal microvascular ECs (639.73 fold, p<0.05) over other human cardiovascular cells like vascular smooth muscle cells, aortic fibroblasts and cardiomyocytes. Severe hypoxia (0.2% O2 for 24h) reduced LINC01235 expression significantly (0.33 fold, p<0.05). SiRNA-mediated LINC01235 silencing in HUVECs (0.12, p<0.05) resulted in decreased proliferation (0.76 fold, p<0.05) and vascular endothelial growth factor A (VEGFA)-stimulated angiogenic sprouting (0.39 fold, p<0.05). Loss of LINC01235 did not affect apoptosis, metabolism or barrier function. Analysis of RNA-sequencing data revealed that many hypoxia-responsive genes were downregulated after knockdown of LINC01235 (siCtrl vs. siLINC01235). These included HIF-3α (0.24 fold, p<5.86e-28) as a potential key regulator of the cellular feedback to hypoxia. Phenotypically, knockdown of HIF3A using siRNAs (0.07 fold, p<0.05) resulted in decreased proliferation (0.82 fold, p<0.05) and VEGFA-stimulated angiogenic sprouting (0.50 fold, p<0.05). Accordingly, hypoxia response and LINC01235 knockdown exhibit a negative correlation based on transcriptomics data (R=−0.157, p<2.2e-16), further emphasizing a role of LINC01235 in hypoxia response. Conclusion In summary, the EC-enriched lncRNA LINC01235 is likely required for the suppression of hypoxia-induced gene expression under normoxic conditions potentially mediated by HIF-3α. Functionally, loss of LINC01235 decreased proliferation and VEGFA-stimulated angiogenic sprouting without an effect on cell death, metabolism or barrier integrity. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): DFG - TRR267

MiR-133a Replacement Attenuates Thoracic Aortic Aneurysm in Mice.

BackgroundThoracic aortic aneurysms (TAAs) occur because of abnormal remodeling of aortic extracellular matrix and are accompanied by the emergence of proteolytically active myofibroblasts. The microRNA miR‐133a regulates cellular phenotypes and is reduced in clinical TAA specimens. This study tested the hypothesis that miR‐133a modulates aortic fibroblast phenotype, and overexpression by lentivirus attenuates the development of TAA in a murine model.Methods and ResultsTAA was induced in mice. Copy number of miR‐133a was reduced in TAA tissue and linear regression analysis confirmed an inverse correlation between aortic diameter and miR‐133a. Analyses of phenotypic markers revealed an mRNA expression profile consistent with myofibroblasts in TAA tissue. Fibroblasts were isolated from the thoracic aortae of mice with/without TAA. When compared with controls, miR‐133a was reduced, migration was increased, adhesion was reduced, and the ability to contract a collagen disk was increased. Overexpression/knockdown of miR‐133a controlled these phenotypes. After TAA induction in mice, a single tail‐vein injection of either miR‐133a overexpression or scrambled sequence (control) lentivirus was performed. Overexpression of miR‐133a attenuated TAA development. The pro‐protein convertase furin was confirmed to be a target of miR‐133a by luciferase reporter assay. Furin was elevated in this murine model of TAA and repressed by miR‐133a replacement in vivo resulting in reduced proteolytic activation.ConclusionsmiR‐133a regulates aortic fibroblast phenotype and over‐expression prevented the development of TAA in a murine model. These findings suggest that stable alterations in aortic fibroblasts are associated with development of TAA and regulation by miR‐133a may lead to a novel therapeutic strategy.

Nuclear mechanosensing drives chromatin remodelling in persistently activated fibroblasts.

Fibrotic disease is caused by the continuous deposition of extracellular matrix by persistently activated fibroblasts (also known as myofibroblasts), even after the resolution of the injury. Using fibroblasts from porcine aortic valves cultured on hydrogels that can be softened via exposure to ultraviolet light, here we show that increased extracellular stiffness activates the fibroblasts, and that cumulative tension on the nuclear membrane and increases in the activity of histone deacetylases transform transiently activated fibroblasts into myofibroblasts displaying condensed chromatin with genome-wide alterations. The condensed structure of the myofibroblasts is associated with cytoskeletal stability, as indicated by the inhibition of chromatin condensation and myofibroblast persistence after detachment of the nucleus from the cytoskeleton via the displacement of endogenous nesprins from the nuclear envelope. We also show that the chromatin structure of myofibroblasts from patients with aortic valve stenosis is more condensed than that of myofibroblasts from healthy donors. Our findings suggest that nuclear mechanosensing drives distinct chromatin signatures in persistently activated fibroblasts.

Cyborg Vessel

Cyborg Vessel

Electronic Blood Vessel

Summary Advances in bioelectronics have great potential to address unsolved biomedical problems in the cardiovascular system. By using poly(L-lactide-co-ϵ-caprolactone), which encapsulates liquid metal to make flexible and biodegradable electrical circuitry, we develop an electronic blood vessel that can integrate flexible electronics with three layers of blood-vessel cells, to mimic and go beyond the natural blood vessel. It can improve the endothelialization process through electrical stimulation and can enable controlled gene delivery into specific parts of the blood vessel via electroporation. The electronic blood vessel has excellent biocompatibility in the vascular system and shows great patency 3 months post-implantation in a rabbit model. The electronic blood vessel would be an ideal platform to enable diagnostics and treatments in the cardiovascular system and can greatly empower personalized medicine by creating a direct link of the vascular tissue-machine interface.

Distinct Redox Signalling following Macrophage Activation Influences Profibrotic Activity

Aims To date, the ROS-generating capacities of macrophages in different activation states have not been thoroughly compared. This study is aimed at determining the nature and levels of ROS generated following stimulation with common activators of M1 and M2 macrophages and investigating the potential for this to impact fibrosis. Results Human primary and THP-1 macrophages were treated with IFN-γ+LPS or IL-4-activating stimuli, and mRNA expression of established M1 (CXCL11, CCR7, IL-1β) and M2 (MRC-1, CCL18, CCL22) markers was used to confirm activation. Superoxide generation was assessed by L-012-enhanced chemiluminescence and was increased in both M(IFN-γ+LPS) and M(IL-4) macrophages, as compared to unpolarised macrophages (MΦ). This signal was attenuated with NOX2 siRNA. Increased expression of the p47phox and p67phox subunits of the NOX2 oxidase complex was evident in M(IFN-γ+LPS) and M(IL-4) macrophages, respectively. Amplex Red and DCF fluorescence assays detected increased hydrogen peroxide generation following stimulation with IL-4, but not IFN-γ+LPS. Coculture with human aortic adventitial fibroblasts revealed that M(IL-4), but not M(IFN-γ+LPS), enhanced fibroblast collagen 1 protein expression. Macrophage pretreatment with the hydrogen peroxide scavenger, PEG-catalase, attenuated this effect. Conclusion We show that superoxide generation is not only enhanced with stimuli associated with M1 macrophage activation but also with the M2 stimulus IL-4. Macrophages activated with IL-4 also exhibited enhanced hydrogen peroxide generation which in turn increased aortic fibroblast collagen production. Thus, M2 macrophage-derived ROS is identified as a potentially important contributor to aortic fibrosis.

Lytic cycle of Besnoitia besnoiti tachyzoites displays similar features in primary bovine endothelial cells and fibroblasts

BackgroundBovine besnoitiosis, caused by the cyst-forming apicomplexan parasite Besnoitia besnoiti, is a chronic and debilitating cattle disease that continues to spread in Europe in the absence of control tools. In this scenario, in vitro culture systems are valuable tools to carry out drug screenings and to unravel host-parasite interactions. However, studies performed in bovine target cells are scarce.MethodsThe objective of the present study was to obtain primary bovine aortic endothelial cells (BAECs) and fibroblast cell cultures, target cells during the acute and the chronic stage of the disease, respectively, from healthy bovine donors. Afterwards, expression of surface (CD31, CD34 and CD44) and intracellular markers (vimentin and cytokeratin) was studied to characterize cell populations by flow cytometry. Next, the lytic cycle of B. besnoiti tachyzoites was studied in both target cells. Invasion rates (IRs) were determined by immunofluorescence at several time points post-infection, and proliferation kinetics were studied by quantitative PCR (qPCR). Finally, the influence of bovine viral diarrhea virus (BVDV) co-infection on the host cell machinery, and consequently on B. besnoiti invasion and proliferation, was investigated in BAECs.ResultsMorphology and cytometry results confirmed the endothelial and fibroblast origins. CD31 was the surface marker that best discriminated between BAECs and fibroblasts, since fibroblasts lacked CD31 labelling. Expression of CD34 was weak in low-passage BAECs and absent in high-passage BAECs and fibroblasts. Positive labelling for CD44, vimentin and cytokeratin was observed in both BAECs and fibroblasts. Regarding the lytic cycle of the parasite, although low invasion rates (approximately 3–4%) were found in both cell culture systems, more invasion was observed in BAECs at 24 and 72 hpi. The proliferation kinetics did not differ between BAECs and fibroblasts. BVDV infection favoured early Besnoitia invasion but there was no difference in tachyzoite yields observed in BVDV-BAECs compared to BAECs.ConclusionsWe have generated and characterized two novel standardized in vitro models for Besnoitia besnoiti infection based on bovine primary target BAECs and fibroblasts, and have shown the relevance of BVDV coinfections, which should be considered in further studies with other cattle pathogens.

Abstract 320: MicroRNA-133a Attenuates the Development of Thoracic Aortic Aneurysm

Rationale: Thoracic aortic aneurysms (TAA) result from dysregulated remodeling of the vascular extracellular matrix, and may occur as result of altered resident cellular phenotype. MicroRNA-133a is reduced in clinical TAA specimens and plays an inhibitory role in regulation of pathological phenotypic switch of vascular cells. Accordingly, this study tested the hypothesis that miR-133a replacement attenuates the development of TAA. Methods and Results: TAA was induced in wild type mice (0.5M CaCl 2 application, 15 minutes). Following 4 weeks, aortic diameter was increased compared to sham operated controls (1094 ± 38 vs 654 ± 8 μm; p<0.05, n=7). Copy number of miR-133a, quantitated by ddPCR, was reduced in TAA tissue (0.28 ± 0.04 vs 0.68 ± 0.11 copy per U6; p<0.05 vs control, n=7). While no change in smooth muscle cell specific Desmin nor α-smooth muscle actin were detected, an increase in copy number of the fibroblast specific discoidin domain receptor 2 (DDR2) and smooth muscle cell myosin heavy chain 11 (MYH11) were observed in TAA tissue. Combined, these results are consistent with the emergence of a population of myofibroblasts with TAA development. Accordingly, fibroblasts were isolated from TAA and sham operated control tissues (n=10). Copy number of miR-133a was reduced in TAA fibroblasts (0.41 ± 0.14 vs 1.60 ± 0.43 copy per U6; p < 0.05 vs control). TAA fibroblast phenotype was compared to controls; adhesion was reduced (-0.85±0.02 vs -0.77±0.02), migration was increased (63±4% vs 47±4%), and rate of collagen disk contraction over 7 hours was increased (-12.2±1.6 vs -8.5±1.0) (all values, p<0.05 vs control). Finally, after TAA induction in mice, a single tail vein injection of either a miR-133a overexpression or scrambled sequence (control) lentivirus was performed. Following 4 weeks, miR-133a replacement attenuated TAA development (800 ± 18 μm vs 1016 ± 16 μm; p < 0.05 vs control; n=8). Conclusion: Aortic fibroblast phenotype is altered during TAA progression, and miR-133a replacement attenuates the development of TAA in a murine model. These unique findings suggest stable alterations in aortic fibroblasts may be associated with pathological extracellular matrix remodeling, and regulation by miR-133a may lead to a novel therapeutic strategy.

Therapeutic Role of Resveratrol and Quercetin on Aortic Fibroblasts of Psammomys obesus After Oxidative Stress by Hydrogen Peroxide.

In our study, we propose to analyze the effects of resveratrol (RES) and quercetin (QRC) on proliferation markers, oxidative stress, apoptosis, and inflammation of aortic fibroblasts of Psammomys obesus after induced oxidative stress by hydrogen peroxide (H2O2). Fibroblasts were incubated in RES 375 μM and QRC 0.083 μM for 24 hours after exposure to H2O2 1.2 mM for 6 hours. We performed the proliferation rate, cells viability, morphological analyses, cytochrome c, Akt, ERK1/2, and p38 MAPK quantification. The redox status was achieved by proportioning of malondialdehyde, nitric monoxide, advanced oxidation protein products, carbonyl proteins, catalase, and superoxide dismutase activity. The inflammation was measured by TNFα, MCP1, and NF-kB assay. The extracellular matrix (ECM) remodeling was performed by SDS-PAGE. Stressed fibroblasts showed a decrease of cell proliferation and viability, hypertrophy and oncosis, chromatin hypercondensation and increase of cytochrome c release characteristic of apoptosis, activation of ERK1/2 and Akt pathway, and decreases in p38 MAPK pathways marking the cellular resistance. The redox state was disrupted by increased malondialdehyde, nitric monoxide, advanced oxidation protein products, carbonyl protein production, catalase and superoxide dismutase activity, and a decreased production of proteins including collagens. Inflammation state was marked by MCP-1, TNFα, and NF-kB increase. Treatment of fibroblasts stressed by RES and QRC inverted the oxidative stress situation decreasing apoptosis and inflammation, and improving the altered redox status and rearrangement of disorders observed in extracellular matrix. H2O2 induced biochemical and morphological alterations leading to apoptosis. An improved general condition is observed after treatment with RES and QRC; this explains the antioxidant and antiapoptotic effects of polyphenols.

Regulation of membrane type-1 matrix metalloproteinase activity and intracellular localization in clinical thoracic aortic aneurysms

ObjectiveMembrane type-1 matrix metalloproteinase (MT1-MMP) is elevated during thoracic aortic aneurysm (TAA) development in mouse models, and plays an important role in the activation of matrix metalloproteinase (MMP)-2 and the release of matrix- bound transforming growth factor-β. In this study, we tested the hypothesis that MT1-MMP is subject to protein kinase C (PKC)–mediated regulation, which alters intracellular trafficking and activity with TAAs. MethodsLevels of MMP-2, native and phosphorylated MT1-MMP, and PKC-δ were measured in aortic tissue from patients with small TAAs (<5 cm; n = 8) and large TAAs (>6.5 cm; n = 8), and compared with values measured in normal controls (n = 8). Cellular localization of green fluorescent protein (GFP)-tagged MT1-MMP was assessed in aortic fibroblasts isolated from control and 4-week TAA mice. The effects of PKC-mediated phosphorylation on MT1-MMP cellular localization and function (active MMP-2 vs phospo-Smad2 abundance) were assessed after treatment with a PKC activator (phorbol-12-myristate-13-acetate [PMA], 100 nM) with and without a PKC-δ–specific inhibitor (röttlerin, 3 μM). ResultsCompared with controls, MT1-MMP abundance was increased in aortas from both TAA groups. Active MMP-2 was increased only in the large TAA group. The abundances of phosphorylated MT1-MMP and activated PKC-δ were enhanced in the small TAA group compared with the large TAA group. MT1-MMP was localized on the plasma membrane in aortic fibroblasts from control mice and in endosomes from TAA mice. Treatment with PMA induced MT1-MMP-GFP internalization, enhanced phospho-Smad2, and reduced MMP-2 activation, whereas röttlerin pretreatment inhibited these effects. ConclusionsPhosphorylation of MT1-MMP mediates its activity through directing cellular localization, shifting its role from MMP-2 activation to intracellular signaling. Thus, targeted inhibition of MT1-MMP may have therapeutic relevance as an approach to attenuating TAA development.

Abstract 25: Inducible Depletion of Calpain-2 Attenuates Angiotensin II-induced Cytoskeletal Structural Protein Destruction During Abdominal Aortic Aneurysm Development in Mice

Background and Objective: In abdominal aortic aneurysm (AAA) patients, structural integrity of the aortic wall is disrupted due to the dissociation of cytoskeletal structural proteins that bridge SMC contractile filaments with extracellular matrix by proteases. However, identity of functional proteases which target cytoskeletal structural linker proteins remain unknown. Recently, using the angiotensin II (AngII)-induced AAA model, we demonstrated that AngII significantly increased fragmentation of cytoskeletal structural protein, Filamin A, in AAAs. Further, using a pharmacological inhibitor and genetic deficient mice, we identified that calpain-2 (a class of calcium-activated, intracellular cysteine proteases) plays a critical role in AngII-induced AAA formation in mice. The purpose of this study was to determine the functional contribution of calpain-2 in AngII-induced cytoskeletal structural protein destruction during AAA development. Methods and Results: Calpain-2 floxed mice that were hemizygous for Cre-ERT2 in an LDLr -/- background were produced by breeding male Cre-ERT2 to female calpain-2 floxed mice. At 8 weeks of age, male Calp-2 x Cre-ERT2 (Cre+) and non-Cre littermates (Cre-) mice were injected with tamoxifen (25 mg/kg, i.p.) daily for 5 consecutive days. After 2 weeks, Western blot analyses showed a complete depletion of calpain-2 protein in the aorta from Cre+ mice compared to Cre- littermates. Male Cre+ and Cre- (N=12 each) mice were fed a Western diet and infused with saline or AngII (1,000 ng/kg/min) by osmotic minipumps for 2 weeks. AngII infusion significantly (P&lt;0.05 vs saline) increased c-terminal fragmentation of cytoskeletal structural protein, talin and kinases such as integrin linked kinase-1 (ILK-1), and focal adhesion kinase in addition to filamin A. Inducible depletion of calpain-2 significantly (P&lt;0.05; Cre+ vs Cre-) blunted AngII-induced fragmentation of talin, filamin A and ILK-1. Further siRNA mediated silencing of calpain-2 in aortic SMCs and fibroblasts significantly reduced AngII-induced fragmentation of filamin A and talin. Conclusion: These findings suggest that calpain-2 plays a critical role in AngII-induced cytoskeletal structural protein fragmentation during AAA development in mice.

Abstract 9487: Implantation of Scaffold-Free Tubular Tissue Using a Bio-3D Printer Augments Vascular Remodeling and Endothelialization

Introduction: Small caliber synthetic vascular grafts are not clinically available. We developed a novel method to create scaffold-free tubular tissue from multicellular spheroids (MCS) using a “Bio-3D printer”-based system, which enables the creation of various three-dimensional structures pre-designed using a computer system. With this system, we created a tubular structure (Fig. 1), and studied its biological features. Methods: We made 1.5 mm in diameter scaffold-free tubular tissues from MCS (1.25 x 10[[Unable to Display Character: &amp;#8311;]] cells) composed of human umbilical vein endothelial cells (40%), human aortic smooth muscle cells (10%) and normal human dermal fibroblasts (50%) using a Bio-3D printer. The vessels were cultured in a perfusion system. We implanted grafts into the abdominal aortas of F344 nude rats, and assessed the flow by ultrasonography and performed histological examinations on the second (N=5) and fifth (N=5) days after implantation. Results: All grafts were patent. Remodeling of the vessel (enlargement of the lumen area and thinning of the wall) was observed (Fig. 2). A layer of endothelial cells was developed after implantation of the graft (Fig. 3). Conclusions: The scaffold-free vascular grafts made of MCS using a Bio-3D printer showed biological features comparable to native vessels. Further studies are warranted toward the clinical application of this novel technology.

Engineering vascular tissue with functional smooth muscle cells derived from human iPS cells and nanofibrous scaffolds

Tissue-engineered blood vessels (TEBVs) are promising in the replacement of diseased vascular tissues. However, it remains a great challenge to obtain a sufficient number of functional smooth muscle cells (SMCs) in a clinical setting to construct patient-specific TEBVs. In addition, it is critical to develop a scaffold to accommodate these cells and retain their functional phenotype for the regeneration of TEBVs. In this study, human induced pluripotent stem cells (iPSCs) were established from primary human aortic fibroblasts, and characterized with the pluripotency markers expression and cells' capabilities to differentiate into all three germ layer cells. A highly efficient method was then developed to induce these human iPSCs into proliferative SMCs. After multiple times of expansion, the expanded SMCs retained the potential to be induced into the functional contractile phenotype of mature SMCs, which was characterized by the contractile response to carbachol treatment, up-regulation of specific collagen genes under transforming growth factor β1 treatment, and up-regulation of specific matrix metalloproteinase genes under cytokine stimulation. We also developed an advanced macroporous and nanofibrous (NF) poly(l-lactic acid) (PLLA) scaffold with suitable pore size and interpore connectivity to seed these human iPSC-derived SMCs and maintain their differentiated phenotype. Subcutaneous implantation of the SMC-scaffold construct in nude mice demonstrated vascular tissue formation, with robust collagenous matrix deposition inside the scaffold and the maintenance of differentiated SMC phenotype. Taken together, this study established an exciting approach towards the construction of patient-specific TEBVs. We established patient-specific human iPSCs, derived proliferative SMCs for expansion, turned on their mature contractile SMC phenotype, and developed an advanced scaffold for these cells to regenerate vascular tissue in vivo.

Abstract 69: Angiotensin II- Canonical TGF-β Signaling Downregulates Apelinergic Pathway in Hypertension

Background: Apelin is a novel peptide which along with its receptor, APJ, mediates apelinergic signaling. Apelinergic signaling plays a critical role in cardiovascular homeostasis, including regulation of blood pressure. While exogenous apelin blocks Angiotensin II (AngII) mediated nuclear signaling, the role and regulation of endogenous apelin in hypertension (HTN) remains obscure. We hypothesize that apelinergic pathway is downregulated in HTN, which is primarily mediated by aberrant AngII signaling. Approach: To test our hypothesis we utilized two mouse models of HTN, including AngII infusion and oral administration of N (ω)-nitro-l-arginine methyl ester (L-NAME). Blood pressure was monitored via noninvasive tail-cuff device. To determine the signaling involved we investigated the effect of AngII on apelinergic pathway in vitro. Results: Cardiac apelin was decreased significantly in both murine models of HTN. Downregulated apelin also corresponded with increased deposition of collagen, and up-regulation of senescence markers including PAI-1. Meanwhile, APJ levels were unaffected in both these hypertensive models. In our in vitro studies AngII downregulated apelin expression in human aortic endothelial cells (HAECs) and human cardiac fibroblasts (HCFs). Furthermore, our studies in AngII infused mice and in HCFs highlight the role of TGF-β-pSMAD signaling, independent of MEK involvement, in AngII induced apelin downregulation. Conclusion and Significance: Our studies demonstrate that aberrant AngII signaling downregulates apelin in HTN. This downregulation involves canonical Tgf-β1 signaling and affects apelin transcription. Importantly, we propose that AngII mediates its hypertensive pathology by decreasing apelinergic regulation. Since exogenous apelin blocks AngII signaling, further knowledge and negation of AngII induced apelin downregulation could result in the development of novel anti-hypertensive therapies.