Smooth Muscle Cell Actin Research Articles

Extracellular glucose triggers metabolic reprogramming of cultured human bronchial epithelial cells and indirect fibroblast activation.

Glucose is essential for energy metabolism, and its usage can determine other cellular functions, depending on the cell type. In some pathological conditions, cells are exposed to high concentrations of glucose for extended periods. In this study, we investigated metabolic, oxidative stress, and cellular senescence pathways in human bronchial epithelial cells (HBECs) cultured in media with physiologically low (5 mm) and high (12.5 mm) glucose concentrations. HBECs exposed to 12.5 mm glucose showed increased glucose routing toward the pentose phosphate pathway, lactate synthesis, and glycogen, but not triglyceride synthesis. These metabolic shifts were not associated with changes in cell proliferation rates, oxidative stress, or cellular senescence pathways. Since hyperglycemia is associated with fibrosis in the lung, we asked whether HBECS could activate fibroblasts. Primary human lung fibroblasts cultured in media conditioned by 12.5 mm glucose-exposed HBECs showed a 1.3-fold increase in the gene expression of COL1A1 and COL1A2, along with twofold increased protein levels of smooth muscle cell actin and 2.4-fold of COL1A1. Consistently, HBECs cultured with 12.5 mm glucose secreted proteins associated with inflammation and fibrosis, such as interleukins IL-1β, IL-10, and IL-13, CC chemokine ligands CCL2 and CCL24, and with extracellular matrix remodeling, such as metalloproteinases (MMP)-1, MMP-3, MMP-9, and MMP-13 and tissue inhibitors of MMPs (TIMP)-1 and -2. This study shows that HBECs undergo metabolic reprogramming and increase the secretion of profibrotic mediators following exposure to high concentrations of glucose, and it contributes to the understanding of the metabolic crosstalk of neighboring cells in diabetes-associated pulmonary fibrosis.

Abstract 2094: Sex Differences In Venous Thrombus Resolution In Mice With Metabolic Syndrome

Introduction: Metabolic syndrome (MS) is highly prevalent and have been shown to be associated with mitochondrial impairment and increased cardiovascular and thrombotic risk. It is less understood how MS impacts venous thrombosis and thrombus resolution and the effect of sex as a biological variable on these processes. We hypothesize that mice with MS will have impaired thrombus resolution, particularly males compared to females. Methods: We used a model of diet-independent MS where wildtype FVB mice that have been born to high fat diet fed mothers exhibit multiple organ manifestations of MS. 29 adult mice underwent surgical induction of deep vein thrombosis (DVT) via inferior vena cava ligation (stasis model) resulting in thrombi formation until day 3 and thrombus resolution until day 7. Immunohistochemistry was used to assess vascular tissue remodeling. Results: Comparing control mice to mice born to obese mothers after DVT, there were no significant differences in thrombus weights on day 3 (1.83±.62 vs 1.48±.53 g/kg, p =.186), suggesting a similar initial thrombus burden. However, mice born to obese mothers had significantly impaired thrombus resolution at day 7 (1.85±.53 vs 1.14±.42 g/kg, Student’s t-test p =.007) as compared to control mice born from mothers fed a regular diet. Importantly, thrombus weights were significantly larger in male offspring of obese mothers as compared to females (2.17±.67 vs 1.43±.26 g/kg, Student’s t test p =.013). Additionally, histology analyses showed associations with increased vein wall collagen deposition (Masson’s trichrome) (25±3 vs 13±4% area stained, Student’s t test, p =.0018) and neovascularization (smooth muscle cell actin) within the thrombi of mice with signs of MS as compared to control mice (70+/-10 vs 40+/-8% area stained, Student’s t test, p =.009). Conclusions: Wildtype mice with diet-independent MS demonstrate diminished thrombus resolution, with males having significantly larger thrombi than females. Impaired DVT resolution was associated with decreased tissue remodeling within the thrombi. These data suggest that MS may induce negative tissue remodeling decreasing thrombus involution and increasing vein wall fibrosis, factors that may increase the likelihood of post thrombotic syndrome.

Comapping Cellular Content and Extracellular Matrix with Hemodynamics in Intact Arterial Tissues Using Scanning Immunofluorescent Multiphoton Microscopy.

Deviation of blood flow from an optimal range is known to be associated with the initiation and progression of vascular pathologies. Important open questions remain about how the abnormal flow drives specific wall changes in pathologies such as cerebral aneurysms where the flow is highly heterogeneous and complex. This knowledge gap precludes the clinical use of readily available flow data to predict outcomes and improve treatment of these diseases. As both flow and the pathological wall changes are spatially heterogeneous, a crucial requirement for progress in this area is a methodology for acquiring and comapping local vascular wall biology data with local hemodynamic data. Here, we developed an imaging pipeline to address this pressing need. A protocol that employs scanning multiphoton microscopy was developed to obtain three-dimensional (3D) datasets for smooth muscle actin, collagen, and elastin in intact vascular specimens. A cluster analysis was introduced to objectively categorize the smooth muscle cells (SMC) across the vascular specimen based on SMC actin density. Finally, direct quantitative comparison of local flow and wall biology in 3D intact specimens was achieved by comapping both heterogeneous SMC data and wall thickness to patient-specific hemodynamic results.

Pharmacological reduction of coagulation factor XI reduces macrophage accumulation and accelerates deep vein thrombosis resolution in a mouse model of venous thrombosis

BackgroundDeep vein thrombosis (DVT) and post‐thrombotic syndrome (PTS) remain highly prevalent despite modern medical therapy. Contact activation is a promising target for safe antithrombotic anticoagulation. The anti‐factor XI (FXI) monoclonal antibody 14E11 reduces circulating levels of FXI without compromising hemostasis. The human recombinant analog, AB023, is in clinical development. The role of FXI in mediation of inflammation during DVT resolution is unknown. ObjectivesInvestigate the effects of pharmacological targeting of FXI with 14E11 in an experimental model of venous thrombosis. MethodsAdult wild‐type CD1 mice were treated with subcutaneous anti‐FXI antibody (14E11, 5 mg/kg) versus saline prior to undergoing surgical constriction of the inferior vena cava (IVC). Mice were evaluated at various time points to assess thrombus weight and volume, as well as histology analysis, ferumoxytol enhanced magnetic resonance imaging (Fe‐MRI), and whole blood flow cytometry. Results14E11‐treated mice had reduced thrombus weights and volumes after IVC constriction on day 7 compared to saline‐treated mice. 14E11 treatment reduced circulating monocytes by flow cytometry and macrophage content within thrombi as evaluated by histologic staining and Fe‐MRI. Collagen deposition was increased at day 3 while CD31 and smooth muscle cell actin expression was increased at day 7 in the thrombi of 14E11‐treated mice compared to saline‐treated mice. ConclusionPharmacologic targeting of FXI enhances the early stages of experimental venous thrombus resolution in wild‐type CD1 mice, and may be of interest for future clinical evaluation of the antibody in DVT and PTS.

SEX SPECIFIC CHANGES OF CAROTID ARTERIAL FUNCTION IN RESPONSE TO VITAMIN D DEFICIENCY

Objective: Male sex and Vitamin D deficiency is associated with increased ischemic stroke risk that may be partially explained by local changes of circulatory control. Our aim was to examine the vascular reactivity and histological changes of isolated carotid artery of female and male rats in Vitamin D deficient and Vitamin D supplemented state. Design and method: Female and male Wistar rats were fed with vitamin D reduced rat chow for eight weeks. Another group received normal chow with further supplementation of vitamin D. Phenylephrine induced contraction, acetylcholine and estrogen induced relaxation of carotid arteries was examined by wire myography. Elastic fiber density was evaluated on resorcin-fuchsin stained tissue sections. Smooth muscle cell actin (SMA), thromboxane A2 receptor and endothelial nitric oxide synthase (eNOS) density and protein tyrosine nitration (NT) was measured by immunohistochemistry. Results: Both male sex and vitamin D deficiency resulted in increased phenylephrine induced contraction. Inhibition of cyclooxygenases by indomethacin increased the degree of contraction only in vitamin D supplemented females. Male gender was associated with reduced acetylcholine induced relaxation, and it was not altered by vitamin D deficiency. Indomethacin increased relaxation in male vessels. Estrogen induced relaxation was significantly reduced by vitamin D deficiency in female but not in male animals. Vitamin D deficiency resulted in reduced elastic fiber density in female, but decreased smooth muscle cell actin density in male rats. Thromboxane A2 receptor density was increased in vitamin D deficient males. NT immunohistochemistry was low in both male group, whereas eNOS was reduced in vitamin D deficient males. Conclusions: In rats male gender was associated with increased phenylephrine induced contraction and reduced acetylcholine induced relaxation of the carotid artery. Vitamin D deficiency caused increased contraction in both sexes; did not change acetylcholine induced relaxation, but reduced the estrogen induced relaxation in female arteries. These functional differences were accompanied with sex specific histological changes of the vessel. The observed sex specific properties and responses of carotid artery to vitamin D deficiency may play a role in the gender difference of cardiovascular and stroke risk.

Sex Differences in Endothelial-to-Mesenchymal Transition in Chronic Thromboembolic Pulmonary Hypertension

<h3>Purpose</h3> Chronic Thromboembolic Pulmonary Hypertension (CTEPH) results from thrombus organization and obstruction of pulmonary arteries. Population studies suggest sex differences in outcomes. Yet, the mechanisms contributing to sex differences in CTEPH remain poorly understood. We propose that endothelial cells present in the organized thrombi undergo endothelial-to-mesenchymal transition (EndoMT); a process in which endothelial cells lose polarity, cell-to-cell contacts, and gradually adopt mesenchymal cells characteristics. Here, we find that markers of EndoMT are significantly higher in female vs male patients, which could contribute to sex differences in CTEPH pathophysiology. <h3>Methods</h3> A retrospective cohort of ten male and ten female CTEPH patients was studied. Pulmonary endarterectomy specimens from these patients were separated into lobar and segmental sections. Two samples (lobar and segmental) from each patient were paraffin embedded and used for Immunohistochemistry (IHC) analysis. IHC staining for markers for EndoMT, including endothelin-1 (ET-1), Transforming growth factor beta-1 (TGF beta-1), vimentin and alpha smooth muscle cell actin (alpha SMA), was performed. Quantitative tissue area analysis for each marker was performed using the HALO image analysis platform. <h3>Results</h3> We found that females have significantly higher total expression of vimentin and TGF beta-1 (Figure A). There was a trend towards increased total expression of ET-1 and alpha SMA in females that is not significantly different from males (Figure B). However, we found that female segmental sections have higher expression levels for vimentin, TGF beta-1, ET-1 and alpha SMA than male segmental sections (Figure C). <h3>Conclusion</h3> Increased expression of EndoMT markers may account for sex differences in the pathophysiology and outcomes of CTEPH. We will validate these findings and further explore EndoMT in the pathogenesis of CTEPH.

Markers of Carotid Plaque Destabilization in Patients With Sleep-Disordered Breathing.

Sleep-disordered breathing (SDB) is a nightly respiratory condition characterized by intermittent hypoxia, leading to oxidative stress, inflammation, and atherosclerosis. However, most cellular markers of human carotid plaques in SDB have not yet been assessed. We aimed at characterizing the cellular, inflammatory, and nitro-oxidative stress markers in carotid plaques obtained from 25 patients undergoing endarterectomy and screened for SDB. Sleep studies were performed during their preoperative hospitalization night using the Watch-PAT 100 device. Oxygen desaturation index (ODI) was used for dividing patients into two groups. Fourteen patients with ODI >5 were designated as SDB and 11 patients with ODI ≤ 5 as non-SDB. Demographics, comorbidities, cardiovascular risk factors, and medications were recorded. Cellular markers in plaques were analyzed by immunofluorescence using confocal microscopy. The expression of neutrophils was identified by CD66b+ and neutrophil elastase, macrophage-foam cells were identified by CD163+, and scavenger receptors by CD68+ and CD36+ expression. Additional markers included 3-nitrotyrosine, endothelial CD31, and smooth muscle cell-actin (SMC-actin). Plaques' lipids were determined by immunohistochemistry with Oil Red O staining. Notably, significantly higher values were found for SDB as compared to patients with non-SDB for 3-nitrotyrosine (p <0.004) and intracellular lipids' content (p <0.02), whereas SMC-actin was lower (p <0.006). There were no significant differences between patients with carotid-associated symptoms (symptomatic) and patients without carotid-associated symptoms (asymptomatic). However, a sub-group of symptomatic patients with co-existent SDB expressed the highest 3-nitrotyrosin, and intracellular lipids levels, and the lowest SMC-actin levels, whereas non-SDB/asymptomatic patients expressed the lowest 3-nitrotyrosin and lipids levels and the highest SMS-actin levels among all patients. Accordingly, ODI was lowest in non-SDB/asymptomatic patients and highest in SDB/symptomatic. In conclusion, plaques of patients with SDB were characterized by markedly increased levels of 3-nitrotyrosine and intracellular lipids content. Conversely, SMC-actin levels were significantly lower. These three markers, such as increased 3-nitrotyrosine and intracellular lipids and decreased SMC-actin are associated with plaque vulnerability and instability. These findings are in line with earlier reports demonstrating increased intima-media thickness in large cohorts of sleep apnea and patients with SDB, and thus, may indicate a higher susceptibility to plaque vulnerability and rapture in patients with SDB.

Sex differences in rat renal arterial responses following exercise training.

During aerobic exercise, hemodynamic alterations occur. Although blood flow in skeletal muscle arteries increases, it decreases in visceral vessels because of mesenterial vasoconstriction. However, maintaining renal blood flow during intensive sport is also a priority. Our aim was to investigate the changes of vascular reactivity and histology of isolated renal artery of male and female rats in response to swim training. Wistar rats were distributed into four groups: male sedentary (MSed), male trained (MTr), female sedentary (FSed), and female trained (FTr). Trained animals underwent a 12-wk-long intensive swimming program. Vascular function of isolated renal artery segments was examined by wire myography. Phenylephrine-induced contraction was lower in FSed than in MSed animals, and it was decreased by training in male but not in female animals. Inhibition of cyclooxygenases by indomethacin reduced contraction in both sedentary groups, and in MTr but not in FTr animals. Inhibition of nitric oxide production increased contraction in both trained groups. Acetylcholine induced relaxation was similar in all experimental groups showing predominant NO-dependency. Elastin and smooth muscle cell actin density was reduced in female rats after aerobic training. This study shows that, as a result of a 12-wk-long training, there are sex differences in renal arterial responses following exercise training. Swimming moderates renal artery vasoconstriction in male animals, whereas it depresses elastic fiber and smooth muscle actin density in females.NEW & NOTEWORTHY We provided the first detailed analysis of the adaptation of the renal artery after aerobic training in male and female rats. As a result of a 12-wk-long training program, the pharmacological responses of renal arteries changed only in male animals. In phenylephrine-induced contraction, cyclooxygenase-mediated vasoconstriction mechanisms lost their significance in female rats, whereas NO-dependent relaxation became a significant contraction reducing factor in both sexes. Early structural changes, such as reduced elastin and smooth muscle cell actin evolves in females.

Tumour-associated Angiogenesis and Intermediate Blood Vessels in Renal Cell Carcinoma.

Renal cell carcinoma is strongly vascularized, and formation of new blood vessels is a complex and multi-step process. In this study, we analysed the subtypes of intermediate blood vessels, as shown by double immunohistochemistry. Tumour-associated blood vessels were identified by double immunostaining based on CD34 and smooth muscle cell actin. Blood vessels were classified both quantitatively and qualitatively based on the expression of the aforementioned two markers. The main criteria to sub-classify intermediate blood vessels was the presence, distribution, and arrangement of perivascular cells. We described three subtypes of intermediate blood vessels found particularly in the tumour area: Subtype 1 lacked perivascular cells, subtype 2 showed scattered pericytes attached to the vascular wall, and subtype 3 showed a continuous layer of perivascular cells on one side. We describe for the first time three subtypes of renal cell carcinoma-associated intermediate blood vessels, which could be important in prognosis and as potential targets for anti-vascular therapy.

The CXCL12/CXCR4 and VEGF165/Nrp1 Pathways Have Differential Effects on Pathological Neo-Angiogenesis and Vascular Remodeling in Hemophilic Arthropathy

Introduction: Joint hypervascularity is characteristic to hemophilic arthropathy (HA), which is a hallmark complication in patients with hemophilia. Angiogenesis in the hemophilic joint is triggered by bleeding and results in excessive vascular remodeling with enlarged irregularly shaped vessels that appear fragile, potentially causing re-bleeding. Therefore, targeting angiogenesis may offer novel treatment avenues in HA. Vascular endothelial growth factor A (VEGF-A) is an important angiogenic mediator implicated in the pathogenesis of HA, but a complete blockade may cause vascular instability with more bleeding, or inhibition of anti-angiogenic isoforms, thereby decreasing therapeutic efficacy. An alternative approach may be to target the co-receptor Neuropilin 1 (Nrp1) that facilitates binding of the pro-angiogenic isoform VEGF165 to its signaling receptor, VEGFR2. Here, we determine whether blocking the binding of VEGF165 to Nrp1 can curb the pathological vascular remodeling associated with hemophilic joint bleeding.Methods and results: Joint bleeding was induced by subpatellar needle puncture in FVIII-/- mice, resulting in pronounced intra- and peri-articular bleeding, evidenced by a drop in hematocrit from ~45% to 28 ± 10% 2 days after injury. The following antibody treatments were initiated 2 days after injury and were administered at 5 mg/kg, twice weekly for 4 weeks: B20-4 targeting murine VEGF-A, and YW107.4.87 blocking the VEGF165 binding site on Nrp1. The CXCR4 antagonist, AMD3100 (300 µg/day by subcutaneous osmotic pump) was included as positive control as it corrects hypervascularity induced by hemophilic joint bleeding (Wyseure et al . ISTH 2017).Four weeks after injury blood flow in the joint, measured by ultrasound-Power Doppler (PD), was increased 2.8-fold in injured vs uninjured FVIII-/- mice (p<0.001; n=13). Anti-VEGF treatment resulted in a similar 3.2-fold increase in PD signal (n=8), indicating that total inhibition of VEGF had no effect on the aberrant blood flow. Inhibition of Nrp1 had a partial effect (1.9-fold increase over baseline; p=0.16 vs no treatment; n=7), whereas inhibition of CXCL12-mediated vascular cell recruitment by the CXCR4 antagonist significantly blunted aberrant PD signals (p<0.05 vs no treatment; n=10). On histology, the total vessel count after anti-Nrp1 treatment was similar to no treatment and significantly greater than baseline (86 anti-Nrp1 vs 80 no treatment vs 42 baseline; p<0.01). Similarly, anti-Nrp1 treatment had no effect on the number of abnormally enlarged vessels (with a diameter ≥ 20 µm) compared to no treatment (15 anti-Nrp1 vs 13 no treatment vs 3 baseline; p<0.01 vs baseline). In contrast, with CXCR4 antagonist treatment both the total vessel count (64; p=0.3 vs baseline) and the number of abnormally enlarged vessels (7; p=0.1 vs baseline) remained low. However, anti-Nrp1 treatment did prevent the 2.5-fold increased perivascular expression of smooth muscle cell actin (αSMA; p<0.01) that typically accompanies the abnormally enlarged vessels in injured FVIII-/- mice. Thus, inhibition of Nrp1 lowered αSMA expression after hemophilic bleeding, similar to baseline and CXCR4 antagonist treatment, although it had no effects on vessel number and enlargement. This suggests that anti-Nrp1 treatment prevents the maturation during vascular remodeling in HA, potentially leaving the vessels destabilized and prone to vascular permeability. Towards that end, staining with Prussian Blue demonstrated increased hemosiderin deposits in anti-Nrp1 treated mice, similar to untreated injured FVIII-/- mice, whereas no signs of macroscopic joint bleeding or hemosiderin deposits were observed after treatment with the CXCR4 antagonist.Conclusions: Abrogating the VEGF165-Nrp1 interaction was not sufficient to prevent neo-angiogenesis and vessel lumen expansion in a murine hemophilic joint injury model. However, inhibition of Nrp1 did reduce perivascular remodeling that may allow regression of the aberrant vasculature over time, but also may promote further vessel instability and additional hemorrhaging. Thus, inhibition of the CXCL12/CXCR4 pathway affects neo-angiogenesis, whereas inhibition of the VEGF165/Nrp1 pathway predominantly affects perivascular maturation, but not neo-angiogenesis. These findings provide novel insights on the potential benefits and pitfalls of anti-angiogenic treatments in HA. Disclosuresvon Drygalski:Hematherix LLC: Membership on an entity's Board of Directors or advisory committees; Pfizer: Honoraria; Novo Nordisk: Honoraria; CSL Behring: Honoraria; Bioverativ: Honoraria; Bayer: Honoraria; Shire: Honoraria. Mosnier:Hematherix LLC: Membership on an entity's Board of Directors or advisory committees.

Disruption of GPR35 Signaling in Bone Marrow-Derived Cells Does Not Influence Vascular Inflammation and Atherosclerosis in Hyperlipidemic Mice.

G-protein-coupled receptor-35 (GPR35) has been identified as a receptor for the tryptophan metabolite kynurenic acid (KynA) and suggested to modulate macrophage polarization in metabolic tissues. Whether GPR35 can influence vascular inflammation and atherosclerosis has however never been tested. Lethally irradiated LdlrKO mice were randomized to receive GPR35KO or wild type (WT) bone marrow transplants and fed a high cholesterol diet for eight weeks to develop atherosclerosis. GPR35KO and WT chimeric mice presented no difference in the size of atherosclerotic lesions in the aortic arch (2.37 ± 0.58% vs. 1.95 ± 0.46%, respectively) or in the aortic roots (14.77 ± 3.33% vs. 11.57 ± 2.49%, respectively). In line with these data, no changes in the percentage of VCAM-1+, IAb + cells, and CD3+ T cells, as well as alpha smooth muscle cell actin expression, was observed between groups. Interestingly, the GPR35KO group presented a small but significant increase in CD68+ macrophage infiltration in the plaque. However, in vitro culture experiments using bone marrow-derived macrophages from both groups indicated that GPR35 plays no role in modulating the secretion of major inflammatory cytokines. Our study indicates that GPR35 expression does not play a direct role in macrophage activation, vascular inflammation, and the development of atherosclerosis.

20-HETE-promoted cerebral blood flow autoregulation is associated with enhanced pericyte contractility

We previously reported that deficiency in 20-HETE or CYP4A impaired the myogenic response and autoregulation of cerebral blood flow (CBF) in rats. The present study demonstrated that CYP4A was coexpressed with alpha-smooth muscle actin (α-SMA) in vascular smooth muscle cells (VSMCs) and most pericytes along parenchymal arteries (PAs) isolated from SD rats. Cell contractile capabilities of cerebral VSMCs and pericytes were reduced with a 20-HETE synthesis inhibitor, HET0016, but restored with 20-HETE analog WIT003. Similarly, intact myogenic responses of the middle cerebral artery and PA of SD rats decreased with HET0016 and were rescued by WIT003. The myogenic response of the PA was abolished in SS and was restored in SS.BN5 and SS.Cyp4a1 rats. HET0016 enhanced CBF and impaired its autoregulation in the surface and deep cortex of SD rats. These results demonstrate that 20-HETE has a direct effect on cerebral mural cell contractility that may play an essential role in controlling cerebral vascular function.

Study on the role of methylation in nonsyndromic cleft lip with or without cleft palate using a monozygotic twin model

ObjectiveThe research on the etiology of nonsyndromic cleft lip with or without cleft palate(NSCL/P) is challenging, and DNA methylation has an impact on the formation of cleft lip and palate. SubjectsIn this study, one of a pair of monozygotic twins (T1) had nonsyndromic cleft lip (NSCL), and one of a pair of monozygotic twins (T2) had nonsyndromic cleft lip and palate (NSCLP). We determined the methylation profiles of more than 850,000 CpGs in the DNA of the blood samples from the two pairs of monozygotic twins. ResultMethylation data indicated that 1184 differentially methylated CpG sites were found in the T1 group (651 hypermethylated and 533 hypomethylated) and 8099 differentially methylated CpG sites in the T2 group (1713 hypermethylated and 6386 hypomethylated) compared with the healthy twin.The common difference was 107 methylation sites.GO enrichment analysis showed that regulation of smooth muscle cell migration and actin cytoskeleton reorganization were the most prominent classes.KEGG pathway enrichment analysis showed that the TGF-β signaling pathway, Notch signaling pathway and Wnt signaling pathway are relevant to the formation of NSCL/P.Two selected genes (NTN1 and PLEKHA7) are involved in the formation of NSCL/P. ConclusionThese findings provide some support for the hypothesis that abnormal DNA methylation may influence the formation of clefts.

Primary soft tissue angiomyolipoma in a dog with unusual clinical features

Angiomyolipoma is an extremely rare neoplasm in animals. It belongs to a group of perivascular epithelioid cell tumours (so-called PEComas). This study reports a case of primary soft tissue angiomyolipoma in a dog with some unusual clinical features. A 4-year old female Labrador dog with a rapidly growing pelvic tumour measuring 30 × 20 cm with a short history of 12 weeks was presented. The tumour was well-circumscribed and pushed into the right vaginal wall and into the perineum. The tumour was completely surgically excised. An extensive histological examination was performed, including immunohistochemical analysis. Histology revealed a mesenchymal neoplasm consisting of three tissue components – mature adipose tissue (which dominated), vessels, and smooth muscle (spindle) cells. The lesion showed positive immunohistochemical staining with smooth muscle actin and desmin in mature-appearing smooth muscle cells and S-100 protein positivity in adipocytes. The diagnosis of angiomyolipoma must be considered in different types of benign and malignant tumours of various lineages. The diagnostic approach to soft tissue tumours, including angiomyolipoma, requires optimal processing and sectioning of resected specimens. This paper is believed to be the first case of primary soft tissue angiomyolipoma reported in the veterinary literature.

Myoepithelial carcinoma of the breast: A rare case presentation

Myoepithelial carcinoma (MC) remains a rarely encountered lesion of the breast. The few cases that have surfaced firmly document the histopathology and immunohistochemistry of this tumor. Here, we present a case of infiltrating MC of the breast in a 44-year-old female who presented with a painful lump. With the subsequent biopsy, the diagnosis was made on the basis of histopathological and immunohistochemistry. Histological examination showed spindle cells with moderate to marked nuclear atypia. Immunohistochemistry showed reactivity in the spindle cells for smooth muscle actin, cytokeratin (AE1/AE3), and p63, indicating a myoepithelial cell lineage of tumor cells. We suggest MCs of the breast be managed with an appropriate surgical clearance. A multidisciplinary approach is usually required.

BMP9 attenuates occurrence of venous malformation by maintaining endothelial quiescence and strengthening vessel walls via SMAD1/5/ID1/α-SMA pathway

Venous malformation (VM) is a type of vascular morphogenic defect in humans with an incidence of 1%. Although gene mutation is considered as the most common cause of VM, the pathogenesis of those without gene mutation remains to be elucidated. Here, we aimed to explore the relation of bone morphogenetic protein 9 (BMP9) and development of VM. At first, we found serum and tissue BMP9 expression in VM patients was significantly lower than that in healthy subjects, detected via enzyme-linked immunosorbent assay. Next, with wound healing assay, transwell assay and tube formation assay, we discovered BMP9 could inhibit migration and enhance tube formation activity of human umbilical vein endothelial cells (HUVECs) via receptor activin receptor-like kinase 1 (ALK1). Besides, BMP9 improved the expression of structural proteins alpha-smooth muscle actin (α-SMA) and Desmin in human umbilical vein smooth muscle cells (HUVSMCs) via activation of the SMAD1/5-ID1 pathway, determined by RNA-based next-generation sequencing, qPCR, immunofluorescence and western blotting. Intriguingly, this effect could be blocked by receptor ALK1 inhibitor, SMAD1/5 inhibitor and siRNAs targeting ID1, verifying the BMP9/ALK1/SMAD1/5/ID1/α-SMA pathway. Meanwhile, knocking out BMP9 in C57BL/6 mice embryo led to α-SMA scarcity in walls of lung and mesenteric vessels, as well as walls of small trachea. BMP9−/− zebrafish also exhibited abnormal vascular maturity, indicating a critical role of BMP9 in vascular maturity and remodeling. Finally, a VM mice model revealed that BMP9 might have therapeutic effect in VM progression. Our study discovered that BMP9 might inhibit the occurrence of VM by strengthening the vessel wall and maintaining endothelium quiescence. These findings provide promising evidences of new therapeutic targets that might be used for the management of VM.

Statin-mediated cholesterol depletion exerts coordinated effects on the alterations in rat vascular smooth muscle cell biomechanics and migration.

This study demonstrates and evaluates the changes in rat vascular smooth muscle cell biomechanics following statin-mediated cholesterol depletion. Evidence is presented to show correlated changes in migration and adhesion of vascular smooth muscle cells to extracellular matrix proteins fibronectin and collagen. Concurrently, integrin α5 expression was enhanced but not integrin α2. Atomic force microscopy analysis provides compelling evidence of coordinated reduction in vascular smooth muscle cell stiffness and actin cytoskeletal orientation in response to statin-mediated cholesterol depletion. Proof is provided that statin-mediated cholesterol depletion remodels total vascular smooth muscle cell cytoskeletal orientation that may additionally participate in altering ex vivo aortic vessel function. It is concluded that statin-mediated cholesterol depletion may coordinate vascular smooth muscle cell migration and adhesion to different extracellular matrix proteins and regulate cellular stiffness and cytoskeletal orientation, thus impacting the biomechanics of the cell. Not only does cholesterol induce an inflammatory response and deposits in foam cells at the atherosclerotic plaque, it also regulates cellular mechanics, proliferation and migration in atherosclerosis progression. Statins are HMG-CoA reductase inhibitors that are known to inhibit cellular cholesterol biosynthesis and are clinically prescribed to patients with hypercholesterolemia or related cardiovascular conditions. Nonetheless, the effect of statin-mediated cholesterol management on cellular biomechanics is not fully understood. In this study, we aimed to assess the effect of fluvastatin-mediated cholesterol management on primary rat vascular smooth muscle cell (VSMC) biomechanics. Real-time measurement of cell adhesion, stiffness, and imaging were performed using atomic force microscopy (AFM). Cellular migration on extra cellular matrix (ECM) protein surfaces was studied by time-lapse imaging. The effect of changes in VSMC biomechanics on aortic function was assessed using an ex vivo myograph system. Fluvastatin-mediated cholesterol depletion (-27.8%) lowered VSMC migration distance on a fibronectin (FN)-coated surface (-14.8%) but not on a type 1 collagen (COL1)-coated surface. VSMC adhesion force to FN (+33%) and integrin α5 expression were enhanced but COL1 adhesion and integrin α2 expression were unchanged upon cholesterol depletion. In addition, VSMC stiffness (-46.6%) and ex vivo aortic ring contraction force (-40.1%) were lowered and VSMC actin cytoskeletal orientation was reduced (-24.5%) following statin-mediated cholesterol depletion. Altogether, it is concluded that statin-mediated cholesterol depletion may coordinate VSMC migration and adhesion to different ECM proteins and regulate cellular stiffness and cytoskeletal orientation, thus impacting the biomechanics of the cell and aortic function.

Tissue-engineered PLLA/gelatine nanofibrous scaffold promoting the phenotypic expression of epithelial and smooth muscle cells for urethral reconstruction.

The repair and regeneration of tissues using tissue-engineered scaffolds represent the ultimate goal of regenerative medicine. Despite rapid developments in the field, urethral tissue engineering methods are still insufficient to replicate natural urethral tissue because the bioactivity of existing scaffolds is inefficient, especially for large tissue defects, which require large tissue-engineered scaffolds. Here, we describe the efficiency of gelatine-functionalized, tubular nanofibrous scaffolds of poly(l-lactic acid) (PLLA) in regulating the phenotypic expression of epithelial cells (ECs) and smooth muscle cells (SMCs) for urethral reconstruction. Flexible PLLA/gelatine tubular nanofibrous scaffolds with hierarchical architecture were fabricated by electrospinning. The PLLA/gelatine nanofibrous scaffold exhibited enhanced hydrophilicity and significantly promoted the adhesion, oriented elongation, and proliferation of New Zealand rabbit autologous ECs and SMCs simultaneously. Compared with pure PLLA nanofibrous scaffold, PLLA/gelatine nanofibrous scaffolds upregulated the expression of keratin (AE1/AE3) in ECs and actin (α-SMA) in SMCs as well as the synthesis of elastin. Three months of in vivo scaffold replacement of New Zealand rabbit urethras indicated that a tubular cellularized PLLA/gelatine nanofibrous scaffold maintained urethral patency and facilitated oriented SMC remodeling, lumen epithelialization, and angiogenesis. Our observations showed the synergistic effects of nano-morphology and biochemical clues in the design of biomimetic scaffolds, which can effectively promote urethral regeneration.

Clinicopathological features of peripheral ossifying fibroma in a series of 41 patients.

Peripheral ossifying fibromas are benign mesenchymal lesions that usually arise in the anterior maxilla of young female patients. Histologically they consist of spindle cell proliferation with focal mineralisation. We reviewed 48 specimens from 41 patients and recorded the clinical data, sex, and age of the patients, site and size of the lesions, treatment, and postoperative outcome. Histologically the presence of mature, woven bone, cementum, and calcifications were evaluated and evaluated immunohistochemically. Lesions were more frequent in female patients in the third and fourth decade, and were usually in the lower maxilla and smaller than 2cm. All lesions were conservatively excised, and they relapsed in eight patients. Histopathologically, the lesions were poorly circumscribed, moderately cellular proliferations, with no discernible architectural pattern. All tumours showed some degree of mineralisation, the presence of immature bone being the most common. Immunohistochemical examination showed staining of tumoural cells for smooth muscle actin and CD68. Lesions tended to occur more commonly in female patients, but one decade later than usually reported. We found a higher recurrence rate in lesions that contained cementum-like material but without bone formation, suggesting a lack of maturation in this group. Immunohistochemical results were consistent with myofibroblastic differentiation but they added no information about the behaviour of the lesions.

Leiomiosarcoma peritoneal en un canino: reporte de caso

El propósito de este estudio fue para presentar y documentar un leiomiosarcoma peritoneal en un canino y sus hallazgos histopatológicos e Inmunohistoquímica. El paciente, una hembra de 5 años de edad, de raza Chow-Chow, fue llevada a la Clínica Veterinaria de la Universidad de los Llanos, debido a pérdida de peso, postración y baja condición corporal. Al examen físico presentó mucosas pálidas, aumento de la silueta abdominal con contenido de fluido serosanguinolento. A la palpación se evidenció en el epigastrio craneal una masa no dolorosa, de consistencia dura, de superficie irregular y desplazable caudalmente. Anormalidades bioquímicas séricas incluyeron proteínas totales plasmáticas aumentadas, globulina y creatinina. Así, una laparotomía exploratoria fue realizada, identificando una masa en el mesenterio unida a la porción de intestino grueso. En el reporte se señalaron los siguientes hallazgos macroscópicos: una masa firme de 18 cm de longitud y 13 cm de ancho, con una superficie irregular, gran vascularización. Las muestras de tejido fueron fijadas en formalina buferada al 10% para análisis histopatológico e Inmunohistoquímica. Las muestras fueron procesadas por los métodos de rutina para microscopía y coloración con hematoxilina-eosina (HE), tricrómico de Masson, anticuerpos anti-citoqueratina CK5, anti-actina de músculo liso, vimentina y KIT(CD117). En conclusión, el tumor fue clasificado por histopatología como de origen mesenquimal e inmunopositividad citoplasmática difusa interna de células neoplásicas para actina de músculo liso, vimentina y negatividad para KIT, condujo al diagnóstico de leiomiosarcoma.