Smooth Muscle Cell Actin Research Articles

The expression of glycophorin A and osteoprotegerin is locally increased in carotid atherosclerotic lesions of symptomatic compared to asymptomatic patients

The aim of this study was to evaluate in detail the histopathological characteristics of endarterectomized carotid atherosclerotic lesions in symptomatic versus asymptomatic patients. Twenty carotid lesions, 10 from asymptomatic and 10 from symptomatic patients who underwent carotid endarterectomy were classified according to histomorphological features. Samples were analyzed for intraplaque localization and for the expression of proteins associated with inflammation, such as CD68, interleukin (IL)-1β, tumor necrosis factor-α (TNF-α), pentraxin-3 (PTX-3), nuclear factor-κB (NF-κB), C-reactive protein (CRP) and transforming growth factor-β (TGF-β), as well as for proteins associated with vascular remodelling, such as matrix-metalloproteinase-9 (MMP-9), glycophorin A (GYPA), osteoprotegerin (OPG), vascular cell adhesion molecule-1 (VCAM-1), endothelin-1 (ET-1), vascular endothelial growth factor (VEGF) and vascular smooth muscle cell actin (VSMA). Corresponding expression scores were compared between the symptomatic and asymptomatic patients and evaluated statistically. The expression of all 14 evaluated markers was significantly elevated in the border zone adjacent to the mixed plaque compared with the unaffected control area of the same sample (p<0,016). The expression scores of GYPA and OPG were significantly higher in the border zones around the calcified (GYPA, p=0.035; OPG, p=0.043) and mixed (GYPA, p<0.001; OPG, p=0.007) plaque zones of symptomatic patients compared to asymptomatic patients. No difference in expression scores was observed for any of the analyzed inflammatory marker proteins between the border zones of symptomatic and asymptomatic patients. In conclusion, the increased expression of GYPA, indicating intraplaque hemorrhage, and OPG, indicating the transdifferentiation of vascular cells, in carotid atherosclerotic lesions may be associated with an increased risk of plaque instability.

Vascular perfusion with fluorescent labeled lectin to study ovarian functions

The aim of this study was to optimize a method to visualize tissue vascularity by perfusing the local vascular bed with a fluorescently labeled lectin, combined with immunofluorescent labeling of selected vascular/tissue markers. Ovaries with the pedicle were obtained from adult non-pregnant ewes. Immediately after collection, the ovarian artery was perfused with phosphate buffered saline (PBS) to remove blood cells, followed by perfusion with PBS containing fluorescently labeled Griffonia (Bandeiraea) simplicifolia (BS1) lectin. Then, half of ovary was fixed in formalin and another half in Carnoy's fixative. BS1 was detected in blood vessels in ovaries fixed in formalin, but not in Carnoy's fixative. Formalin fixed tissue was used for immunofluorescence staining of two markers of tissue function and/or structure, Ki67 and smooth muscle cell actin (SMCA). Ki67 was detected in granulosa and theca cells, luteal and stromal tissue, and a portion of Ki67 staining was co-localized with blood vessels. SMCA was detected in pericytes within the capillary system, in blood vessels in all ovarian compartments, and in the stroma. Thus, blood vessel perfusion with fluorescently labeled lectin combined with immunohistochemistry, microscopy, and imaging techniques provide an excellent tool to study angiogenesis, vascular architecture, and organ structures and function in physiological and pathological conditions.

Down-regulation of expression of α- smooth muscle actin in airway smooth muscle cells by allicin and its mechanisms

To investigate the effect of allicin on the expression of α-smooth muscle actin (α-SMA) in airway smooth muscle cells (ASMC), and to evaluate the mechanism of allicin on inhibition of airway remodeling. The human ASMCs were treated for 48 hours with 0, 2.5, 5.0, and 10.0 μmol/L allicin, respectively, and the total RNA and protein of the cells were collected. The mRNA expression of α-SMA was determined by real-time polymerase chain reaction (PCR) analysis. The protein expression of α-SMA and phosphorylation Smad1 (p-Smad1) were assessed by Western blotting analysis. After the treatment with allicin for 48 hours in a dose of 0, 2.5, 5.0, and 10.0 μmol/L respectively, the mRNA expression of α-SMA was down-regulated (0.543±0.121, 0.354±0.072, 0.223±0.058, and 0.191±0.034, respectively), with statistically significant difference among groups (all P<0.05), and the protein expression of α-SMA and p-Smad1 was also gradually down-regulated in a dose-dependent manner [α-SMA protein (grad value ratio): 0.96±0.02, 0.72±0.16, 0.54±0.11, and 0.31±0.14, respectively; p-Smad1 protein (grad value ratio): 0.94±0.03, 0.76±0.13, 0.62±0.11, and 0.43±0.12, respectively), with statistically significant difference among groups (all P<0.05). Allicin depresses the mRNA and protein expression of α-SMA, and inhibits p-Smad1 in a dose-dependent manner, thus interrupts the transforming growth factor-β and Rho kinase signal pathway.

Abstract 18255: MicroRNA Analysis in Experimental Models of Vein Graft Neointima Formation Indentifies a Critical Role For Mir-21 in Neointima Formation

Background: Coronary artery bypass grafting (CABG) is an established treatment to relieve the symptoms of ischemic heart disease. However premature graft failure, in saphenous veins is a major limitation. One of the principal etiological factors responsible for vein graft failure is activation and phenotypic modulation of smooth muscle cells (SMC), resulting in accelerated rates of proliferation and migration. MicroRNAs (miRNAs) are short, single stranded, non-coding RNAs that play important roles in modulating cell phenotypes through regulation of gene expression. Methods and results: In order to assess the expression levels of miR-21 in the progression of vein graft neointimal formation we quantified miR-21 levels in three well-established models of vein graft disease. These were the in vivo porcine model of interposition graft, the isogenic mouse model, as well as the ex vivo human sapehnous vein model of neointimal formation. MiR-21 levels were up-regulated approximately 7-fold in both the porcine and murine models at 7 and 28 days following grafting (n=6). In surgically prepared HSV which were subjected to culture ex vivo we found that miR-21 levels were elevated approximately 3-fold following 7 and 14 days of culture (n=6). In situ localization in the HSV demonstrated that miR-21 was expressed most intensely in the neointimal layer of HSV which also stained positive for SMC actin. Co-localization studies in murine vein grafts revealed that miR-21 was localised to areas of the graft that stained positive for proliferating cell nuclear antigen with substantial co-staining with SMC actin. We assessed the potential functional role of miR-21 in vein graft failure using miR-21 knockout mice. Analysis of neointimal lesion size in miR-21 knockout mice revealed an 81% reduction in neointimal area compared to wild type controls (p&lt; 0.0001, n=7-10/group). Conclusion: These findings demonstrate that miR-21 is up-regulated in multiple models of vein graft disease and that genetic abolition of miR-21 levels can attenuate neointimal formation in response to grafting. Therein, modulation of miR-21 expression represents a novel therapeutic opportunity in the setting of vein graft neoinitma formation.

Circulation: Clinical Summaries

<i>Circulation:</i> Clinical Summaries

Phosphodiesterase Type 5 Inhibition Reverts Prostate Fibroblast-to-Myofibroblast Trans-Differentiation

Phosphodiesterase type 5 (PDE5) inhibitors have been demonstrated to improve lower urinary tract symptoms secondary to benign prostatic hyperplasia (BPH). Because BPH is primarily driven by fibroblast-to-myofibroblast trans-differentiation, this study aimed to evaluate the potential of the PDE5 inhibitor vardenafil to inhibit and reverse trans-differentiation of primary human prostatic stromal cells (PrSC). Vardenafil, sodium nitroprusside, lentiviral-delivered short hairpin RNA-mediated PDE5 knockdown, sodium orthovanadate, and inhibitors of MAPK kinase, protein kinase G, Ras homolog family member (Rho) A, RhoA/Rho kinase, phosphatidylinositol 3 kinase and protein kinase B (AKT) were applied to PrSC treated with basic fibroblast growth factor (fibroblasts) or TGFβ1 (myofibroblasts) in vitro, in chicken chorioallantoic membrane xenografts in vivo, and to prostatic organoids ex vivo. Fibroblast-to-myofibroblast trans-differentiation was monitored by smooth muscle cell actin and IGF binding protein 3 mRNA and protein levels. Vardenafil significantly attenuated TGFβ1-induced PrSC trans-differentiation in vitro and in chorioallantoic membrane xenografts. Enhancement of nitric oxide/cyclic guanosine monophosphate signaling by vardenafil, sodium nitroprusside, or PDE5 knockdown reduced smooth muscle cell actin and IGF binding protein 3 mRNA and protein levels and restored fibroblast-like morphology in trans-differentiated myofibroblast. This reversal of trans-differentiation was not affected by MAPK kinase, protein kinase G, RhoA, or RhoA/Rho kinase inhibition, but vardenafil attenuated phospho-AKT levels in myofibroblasts. Consistently, phosphatidylinositol 3 kinase or AKT inhibition induced reversal of trans-differentiation, whereas the tyrosine phosphatase inhibitor sodium orthovanadate abrogated the effect of vardenafil. Treatment of prostatic organoids with vardenafil ex vivo reduced expression of myofibroblast markers, indicating reverse remodeling of stroma towards a desired higher fibroblast/myofibroblast ratio. Thus, enhancement of the nitric oxide/cyclic guanosine monophosphate signaling pathway by vardenafil attenuates and reverts fibroblast-to-myofibroblast trans-differentiation, hypothesizing that BPH patients might benefit from long-term therapy with PDE5 inhibitors.

Characterization of fibrosis-promoting factors and siRNA-mediated therapies in C-protein-induced experimental autoimmune myocarditis

Due to poor proliferation abilities of cardiomyocytes, the repair process in the heart after insults is often associated with fibrosis formation. In this study, we characterized inflammation and/or fibrosis-related molecules in the heart with experimental autoimmune carditis. Immunohistochemical examinations reveled that expression of tenascin-C (TNC), matrix metalloproteinase-9 (MMP-9), transforming growth factorβ1 (TGF-β1), connective tissue growth factor (CTGF) and α smooth muscle cell actin (αSMA) peaked at 2weeks post-immunization but only TGF-β1 expression was sustained at 8weeks. Administration of siRNAs for MMP-2 (siMMP-2) and for MMP-9 (siMMP-9) alone did not modulate inflammation and fibrosis. In contrast, simultaneous administration of siMMP-2 and siMMP-9 significantly reduced inflammation and fibrosis. Of note, siRNA treatment for TGF-β1, which is an anti-inflammatory cytokine, increased inflammation and decreased fibrosis. These findings suggest that in case of diseases characterized by initial inflammation and subsequent fibrosis, immunotherapies should target inflammation, not fibrosis, because the latter therapies exacerbate inflammation.

High-Resolution [18]Fluorodeoxyglucose-Positron Emission Tomography and Coregistered Magnetic Resonance Imaging of Atherosclerotic Plaque From a Patient Undergoing Carotid Endarterectomy

In this Institutional Review Board-approved investigation, an 82-year-old male underwent carotid endarterectomy for a >80% asymptomatic carotid stenosis. Six weeks prior, the subject had undergone carotid endarterectomy of the contralateral carotid artery for a symptomatic >70% stenosis. Approximately 1 hour before surgery, the patient was injected with 7.9 mCi [18]Fluorodeoxyglucose (FDG). After resection of the plaque using a surgical technique that maintained its integrity, the specimen was imaged using micro-positron emission tomography (PET) (Siemens Inveon) and magnetic resonance imaging (3T Philips Achieva). Subsequently, the specimen was fixed in formalin, sectioned at 1 mm intervals, and stained with hematoxylin and eosin. Immunocytochemistry was performed using antibodies to detect macrophages (HAM-56, Dako, 1:100), leukocytes (CD-45, Dako, 1:200), and smooth muscle cell actin (anti–σ-actin Sigma, 1:100). The …

Abstract 58: Intratracheal Delivery of Autologous Bone Marrow-derived Cells Attenuate Muscularization and Inflammation in Lungs of Monocrotaline-induced Pulmonary Hypertension Model Rats.

Background: Vascular remodeling and inflammation contribute to the pathogenesis and/or development of pulmonary hypertension. Bone marrow-derived cells (BMCs) include mesenchymal stem cells and progenitor cells play critical role in tissue repair as well as anti-inflammatory effects. Aim of this study was to investigate the effect of intratracheal delivered BMCs on monocrotaline (MCT)-induced muscuralization and inflammation in rat lung. METHODS: BMCs were isolated and cultured for 3 weeks. Seven days following MCT (60 mg/kg) treatment, DiI labeled autologous BMCs were intratracheally delivered to male Sprague-Dawley rats. Twenty-eight days following the MCT treatment, rats had hemodynamic studies. Hearts and lungs were removed to evaluate right ventricular (RV) hypertrophy and lung pathologies. Results: MCT treated rats had increased RV systolic pressure (RVSP), RV weight as well as wall thickness of small pulmonary arteries and alveolar septum thickness than those of placebo-treated control rats (all p values &lt;0.01). Intratracheal delivery of autologous BMCs (1x106) 7 days after MCT prevented the increase in RVSP at 4 weeks compared with MCT alone (45.1 ± 2.23 versus 35.0 ± 3.84 mmHg, respectively; p&lt;0.05). Furthermore, the intratracheal delivery of BMCs significantly attenuated the pulmonary vessel wall thickness (54.4 % ± 2.25 versus 17.8 % ± 2.69, p&lt;0.01), and alveolar septum thickening (155.1 % ± 1.56 versus 114.2 % ± 1.16, p&lt;0.01) accompanied with decreased numbers of inflammatory cells in perivascular areas and expression of proinflammatory cytokines compared with MCT alone. Immunohistochemical studies showed monocyte-like shaped DiI positive cells distributed in alveolar areas in BMCs delivered lungs, and some of those DiI positive cells expressed CD 68 and alpha smooth muscle cell actin. Conclusion: Intratracheal delivery of autologous BMCs prevented MCT-induced pulmonary hypertension by attenuation of vascular muscularization and inflammation in rat lung.

Histopathologic, immunohistochemical and ultrastructural features of a granular cell tumour in an Australian parakeet (Melopsittacus undulatus)

An adult male Australian parakeet (Melopsittacus undulatus) presented a firm nodular lesion in the lateral metacarpal region of the right wing. Microscopically, there were neoplastic cells, round and polyhedral in shape, with abundant, slightly eosinophilic granular cytoplasm; they were strongly periodic-acid Schiff-positive and resistant to diastase digestion. Some groups of neoplastic cells were immunopositive for smooth muscle actin and desmin. There was no immunopositivity for S-100 protein, CD68 and cytokeratin. Ultrastructurally, the neoplastic cells were round and polygonal in shape, and they were characterized by abundant cytoplasm with numerous homogeneous osmophilic bodies covered by an electron-dense membrane (lysosomes). The histopathologic, immunohistochemical and ultrastructural features of the neoplastic tissue are consistent with a granular cell tumour, which has been described in different animal species and anatomic locations; however, this seems to be an infrequent neoplasm in Australian parakeets. The immunopositivity of the neoplastic cells for smooth muscle actin and desmin, as well as slight positivity for muscle with Masson's trichrome, suggest that this is a tumour of myogenic origin.

Abstract 360: Development of a Human Tissue-Engineered Blood Vessel from Adipose-Derived Stem Cells

There exists a clear need for alternative sources of small-diameter vascular grafts for treating the millions of patients who suffer from cardiovascular disease each year. Bypass surgery or replacement of defective vessels is often required to treat coronary heart disease, but there is a limited supply of suitable autologous grafts, and synthetic grafts are ineffective for replacement of small-diameter vessels. Inherent thrombogenicity, compliance mismatch, and limited patency rates are all complications with current options. Tissue engineering has the potential to overcome these limitations by producing a readily-available vascular graft completely from biological material. It is the objective of this study to fabricate such a small-diameter tissue engineered blood vessel (TEBV) by using the human amniotic membrane as a mechanically-sound biological substrate. Our technology begins by differentiating adipose-derived stem cells into smooth muscle cells (SMCs) and seeding them onto a flat sheet of the amniotic membrane. We assessed our hypothesis that several types of SMCs can successfully attach and proliferate on this membrane by fluorescently staining cell nuclei with DAPI and characteristic SMC actin filaments with phallotoxins. After 7 days in static culture, the cell-seeded sheet was wrapped around a 3mm O.D. removable mandrel with 6-7 revolutions to develop a tubular construct with architecture akin to that of a muscular artery’s tunica media layer. After a 2 week static culture period, the TEBV was characterized for its biochemical and mechanical properties. We examined the contraction of the vessel in response to carbachol, a specific agonist for SMCs, and compared our results with the contraction of porcine coronary arteries. Burst pressure and elastic modulus tests were also performed. The mechanical integrity of this construct can be further improved upon its exposure to appropriate physiological conditions in a perfusion bioreactor. We show that adipose-derived endothelial cells (ECs) can be also be seeded into the lumen of this construct to prevent platelet adhesion. In conclusion, we have developed a small-diameter TEBV with off-the-shelf availability using a completely biological material seeded with patient-own stem cells.

ET-1 deletion from endothelial cells protects the kidney during the extension phase of ischemia/reperfusion injury

BackgroundThe prognosis of patients after acute kidney injury (AKI) is poor and treatment is limited. AKI is mainly caused by renal ischemia/reperfusion injury (IRI). During the extension phase of IRI, endothelial damage may participate in ischemia and inflammation. Endothelin-1 (ET-1) which is mostly secreted by endothelial cells is an important actor of IRI, particularly through its strong vasoconstrictive properties. We aimed to analyze the specific role of ET-1 from the endothelial cells in AKI. MethodsWe used mice lacking ET-1 in the vascular endothelial cells (VEETKO). We induced IRI in VEETKO mice and wild type controls by clamping both kidneys for 30min. Sham operated mice were used as controls. Mice were sacrificed one day after IRI in order to investigate the extension phase of IRI. Kidney function was assessed based on serum creatinine concentration. Levels of expression of ET-1, its receptor ETA, protein kinase C, eNOS, E-Cadherin and inflammation markers were evaluated by real time PCR or western blot. Tubular injury was scored on periodic acid Schiff stained kidney preparations. Lumen and wall area of small intrarenal arteries were measured on kidney slices stained for alpha smooth muscle cell actin. Oxidative stress, macrophage infiltration and cell proliferation was evaluated on slices stained for 8-hydroxy-2′-deoxyguanosine, F4/80 and PCNA, respectively. ResultsIRI induced kidney failure and increased ET-1 and ETA receptor expression. This was accompanied by tubular injury, wall thickening and reduction of lumen area/wall area ratio of small renal arteries, increased oxidative stress and inflammation. These parameters were attenuated in VEETKO mice. ConclusionOur results suggest that suppression of ET-1 from the endothelial cells attenuates IRI kidney injury. Blocking ET-1 effects may represent a therapeutic strategy in the management of AKI.

Nitrite anion stimulates ischemic arteriogenesis involving NO metabolism

Nitric oxide (NO) is a potential regulator of ischemic vascular remodeling, and as such therapies augmenting its bioavailability may be useful for the treatment of ischemic tissue diseases. Here we examine the effect of administering the NO prodrug sodium nitrite on arteriogenesis activity during established tissue ischemia. Chronic hindlimb ischemia was induced by permanent unilateral femoral artery and vein ligation. Five days postligation; animals were randomized to control PBS or sodium nitrite (165 μg/kg) therapy twice daily. In situ vascular remodeling was measured longitudinally using SPY angiography and Microfil vascular casting. Delayed sodium nitrite therapy rapidly increased ischemic limb arterial vessel diameter and branching in a NO-dependent manner. SPY imaging angiography over time showed that nitrite therapy enhanced ischemic gracillis collateral vessel formation from the profunda femoris to the saphenous artery. Immunofluorescent staining of smooth muscle cell actin also confirmed that sodium nitrite therapy increased arteriogenesis in a NO-dependent manner. The NO prodrug sodium nitrite significantly increases arteriogenesis and reperfusion of established severe chronic tissue ischemia.

Abstract 1480: Regulation of ovarian tumor growth and stromal cell infiltration by the HU177 biomechanical ECM switch

Abstract Accumulating evidence indicates that the malignant behavior of tumors depends not only on tumor cells themselves, but also on the stromal cells that comprise the malignant tumor mass. Experimental findings suggest that stromal cells such as cancer-associated fibroblast (CAF) may play important roles in promoting tumor growth and metastasis as well as regulating the efficacy of certain chemotherapeutic drugs. However, developing novel clinical strategies that selectively and simultaneously impacts tumor and stromal cells remains challenging. Alterations in the integrity and molecular composition of the extracellular matrix (ECM) are hallmarks of tumor progression. Our previous studies have shown that structural remodeling of the ECM can result in localized triggering of what we have termed “biomechanical ECM switches” or changes in the three-dimensional structure of pre-existing ECM molecules. A humanized antibody (TRC093/D93) specifically directed to the HU177 cryptic collagen epitope that is selectively exposed following triggering of a biomechanical ECM switch has been developed, and a human phase-I clinical trial was recently completed with encouraging results. Here we provide evidence that the HU177 biomechanical ECM switch is triggered within human ovarian tumors resulting in the exposure of the HU177 cryptic collagen epitope. The relative exposure of the HU177 cryptic site was significantly (P&amp;lt;0.05) enhanced in biopsies of malignant ovarian tumors as compared to benign ovarian lesions. Selective targeting of the HU177 cryptic collagen epitope by Mab D93 significantly (P&amp;lt;0.05) inhibited SKOV-3 tumor growth by approximately 70% as compared to controls. Tumors from these mice exhibited reduced angiogenesis, elevated levels of apoptosis and a significant reduction in infiltration of alpha smooth muscle cell actin (αSMC-actin) positive stromal fibroblasts. Importantly, while Mab D93 inhibited SKOV-3 tumor cell adhesion to denatured collagen type-I and enhanced the expression of the cyclin dependent kinase inhibitor P27KIP1, it also selectively inhibited (80%) fibroblast migration on denatured collagen type-I that was induced by either FGF-2 or SKOV-3 condition medium. Collectively these studies provide evidence that specific targeting of the HU177 cryptic collagen epitope may represent a highly selective strategy to inhibit ovarian tumor growth in part by limiting stromal cell invasion. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1480. doi:1538-7445.AM2012-1480

Osteopontin regulates α‐smooth muscle actin and calponin in vascular smooth muscle cells

vSMCs (vascular smooth muscle cells) lose differentiation markers and gain uncontrolled proliferative activity during the early stages of atherosclerosis. Previous studies have shown that OPN (osteopontin) mRNA and protein levels increase significantly on induction of proliferative activity by allylamine (an atherogenic amine) and that this response can be inhibited by OPN antibodies. We have investigated the role of OPN in vSMC differentiation. Primary cultures of aortic mouse vSMCs were transfected with an OPN expression plasmid and several vSMC differentiation markers including α-SM actin (α-smooth muscle actin), SM22-α, tropomyosin and calponin were monitored in this cellular model. α-SM actin and calponin protein levels were significantly decreased by OPN overexpression. Down-regulation of α-SM actin and calponin was also observed on extracellular treatment of mouse vSMCs with recombinant OPN. In addition, calponin mRNA was significantly decreased under serum-restricted conditions when OPN mRNA was dramatically increased, while α-SM actin mRNA remained unchanged. These data indicate that OPN down-regulates α-SM actin and calponin expression through an extracellular signalling pathway. Functional connectivity between OPN and vSMC differentiation markers has been established. Since vSMCs lose differentiation features during early atherosclerosis, a mechanistic basis for OPN functions as a critical regulator of proliferative cardiovascular disease has been presented.

Adenovirus-mediated E2-EPF UCP gene transfer prevents autoamputation in a mouse model of hindlimb ischemia.

E2-EPF ubiquitin carrier protein (UCP) stabilizes hypoxia-inducible factor-1α (HIF-1α) inducing ischemic vascular responses. Here, we investigated the effect of UCP gene transfer on therapeutic angiogenesis. Adenovirus-encoded UCP (Ad-F-UCP) increased the expression of vascular endothelial growth factor (VEGF) and fibroblast growth factor-2 (FGF-2) in cells and mice. Conditioned media from UCP-overexpressing cells promoted proliferation, tubule formation, and invasion of human umbilical-vascular-endothelial cells (HUVECs), and vascularization in chorioallantoic membrane (CAM) assay. Ad-F-UCP increased the vessel density in the Martigel plug assay, and generated copious vessel-like structures in the explanted muscle. The UCP effect on angiogenesis was dependent on VEGF and FGF-2. In mouse hindlimb ischemia model (N = 30/group), autoamputation (limb loss) occurred in 87% and 68% of the mice with saline and Ad encoding β-galactosidase (Ad-LacZ), respectively, whereas only 23% of the mice injected with Ad-F-UCP showed autoamputation after 21 days of treatment. Ad-F-UCP increased protein levels of HIF-1α, platelet-endothelial cell adhesion molecule-1 (PECAM-1), smooth muscle cell actin (SMA) in the ischemic muscle, and augmented blood vessels doubly positive for PECAM-1 and SMA. Consequently, UCP gene transfer prevented muscle degeneration and autoamputation of ischemic limb. The results suggest that E2-EPF UCP may be a target for therapeutic angiogenesis.

Three-Dimensional Microstructural Changes in Murine Abdominal Aortic Aneurysms Quantified Using Immunofluorescent Array Tomography

This study investigated the spatial and temporal remodeling of blood vessel wall microarchitecture and cellular morphology during abdominal aortic aneurysm (AAA) development using immunofluorescent array tomography (IAT), a high-resolution three-dimensional (3D) microscopy technology, in the murine model. Infrarenal aortas of C57BL6 mice (N=20) were evaluated at 0, 7, and 28 days after elastase or heat-inactivated elastase perfusion. Custom algorithms quantified volume fractions (VF) of elastin, smooth muscle cell (SMC) actin, and adventitial collagen type I, as well as elastin thickness, elastin fragmentation, non-adventitial wall thickness, and nuclei amount. The 3D renderings depicted elastin and collagen type I degradation and SMC morphological changes. Elastin VF decreased 37.5% (p<0.01), thickness decreased 48.9%, and fragmentation increased 449.7% (p<0.001) over 28 days. SMC actin VF decreased 78.3% (p<0.001) from days 0 to 7 and increased 139.7% (p<0.05) from days 7 to 28. Non-adventitial wall thickness increased 61.1%, medial nuclei amount increased 159.1% (p<0.01), and adventitial collagen type I VF decreased 64.1% (p<0.001) over 28 days. IAT and custom image analysis algorithms have enabled robust quantification of vessel wall content, microstructure, and organization to help elucidate the dynamics of vascular remodeling during AAA development.

Expression of angiogenic growth factors and structural proteins in Kazakh patients with ruptured and unruptured cerebral aneurysms in Xinjiang

Objective To evaluate differences in the expression of angiogenic growth factors( VEGF and TGF- a) and structural proteins (Collagen Ⅲ,Collagen Ⅳ,smooth muscle cell actin ) in Kazakh patients with ruptured and unruptured cerebral aneurysms in Xinjiang,to discuss the possible mechanism of cerebral aneurysm growth and rupture.Method Twelve paired specimens( one ruptured and the other one was unruptured in same patient,total 24 specimens) of cerebral aneurysms which of them obtained during the surgery for Kazakh patients with multiple cerebral aneurysms and the five specimen from the cadavers were immunohistologically stained for angiogenic growth factors( VEGF and TGF - a) and structural proteins (Collagen Ⅲ,Collagen Ⅳ and smooth muscle cell actin).Results VEGF was not expressed in normal arterial walls,while expression was weak in unruptured ones and less significant in ruptured ones.TGF - a was expressed significantly in normal arterial walls,expressed less significantly in uuruptured aneurysm walls,and expression was weak in ruptured ones.Type Ⅲ and Type Ⅳ Collagen were expressed significantly in the normal arterial walls,expression was became less significant in unruptured aneurysm walls and expression was weak in ruptured ones.Smooth muscle cell actin was expressed significantly in the normal arterial walls,expression became less significant in unruptured aneurysm walls and expression was weak in ruptured ones.Conclusions Abnormal expression of angiogenic growth factors and degradation of structural proteins are one of the key mechanisms of cerebral aneurysm formation and rupture. Key words: Intracranial aneurysm; Angiogenic factors; Structural proteins

Spice up the hypertension diet - curcumin and piperine prevent remodeling of aorta in experimental L-NAME induced hypertension

BackgroundIncrease of blood pressure is accompanied by functional and morphological changes in the vascular wall. The presented study explored the effects of curcuma and black pepper compounds on increased blood pressure and remodeling of aorta in the rat model of experimental NO-deficient hypertension.MethodsWistar rats were administered for 6 weeks clear water or L-NAME (40 mg/kg/day) dissolved in water, piperine (20 mg/kg/day), curcumin (100 mg/kg/day) or their combination in corn oil by oral gavage. The systolic blood pressure was measured weekly. Histological slices of thoracic aorta were stained with hematoxylin and eosin, Mallory's phosphotungstic acid hematoxylin (PTAH), orcein, picrosirius red and van Gieson staining and with antibodies against smooth muscle cells actin. Microscopic pictures were digitally processed and morphometrically evaluated.ResultsThe increase of blood pressure caused by L-NAME was partially prevented by piperine and curcumin, but the effect of their combination was less significant. Animals with hypertension had increased wall thickness and cross-sectional area of the aorta, accompanied by relative increase of PTAH positive myofibrils and decrease of elastin, collagen and actin content. Piperine was able to decrease the content of myofibrils and slightly increase actin, while curcumin also prevented elastin decrease. The combination of spices had similar effects on aortic morphology as curcumin itself.ConclusionsAdministration of piperine or curcumin, less their combination, is able to partially prevent the increase of blood pressure caused by chronic L-NAME administration. The spices modify the remodeling of the wall of the aorta induced by hypertension. Our results show that independent administration of curcumin is more effective in preventing negative changes in blood vessel morphology accompanying hypertensive disease.

Effect of Chronic High Altitude Hypoxia on Foetal and Maternal Juxta-Alveolar Distal Pulmonary Smooth Muscle Cells Actin and Calponin Organisation and Growth Profiles

The effect of chronic high altitude hypoxia (CHAH) in the juxta-alveolar region near the air-blood interface is unknown because of the experimental inaccessibility of this region. To examine primary cultures of digested juxta-alveolar smooth muscle cells for hypoxia-induced changes. Smooth Muscle Cells (SMCs) obtained by dispase digestion of the extreme lung parenchyma were used to study the effect of CHAH in the juxta-alveolar region and foetal and maternal cells were compared. Pulmonary venous SMCs were also obtained from dissected 5th to 7th generation levels pulmonary veins (<0.5 mm). Fluorescence tagged antibodies against alpha smooth muscle actin (alpha SMA) and calponin respectively were used as markers to identify cellular structural differences by routine immunohistochemistry. Comparison of the functional integrity of the cells was made using their growth profiles obtained by radiolabeled thymidine incorporation and liquid scintillation counting. Marked differences were seen in juxta-alveolar SMCs obtained by digestion of extreme lung parenchyma of hypoxic sheep. Hypoxic adult sheep cells showed increased filamentation. Hypoxic foetal sheep cells showed internal restructuring and disorganization of both alpha-SMA and calponin filaments. The growth profiles of juxta-alveolar SMCs showed that the hypoxia-affected cells of both the foetus and adult sheep had a fast initial growth rate peaking at 48h while their normoxic equivalents had a steadier growth rate peaking at 72h. Hypoxia-affected cells showed contact inhibition at ~50% subconfluence and apoptosis by 48h. Chronic high altitude hypoxia causes both phenotypical and functional changes in pulmonary smooth muscle cells near the air/blood interface.