Qilin Ao

Transdifferentiation of pulmonary arteriolar endothelial cells into smooth muscle-like cells regulated by myocardin involved in hypoxia-induced pulmonary vascular remodelling.

Myocardin gene has been identified as a master regulator of smooth muscle cell differentiation. Smooth muscle cells play a critical role in the pathogenesis of hypoxia‐induced pulmonary hypertension (PH) and pulmonary vascular remodelling (PVR). The purpose of this study was to investigate the change of myocardin gene expression in the pulmonary vessels of hypoxia‐induced PH affected by Sildenafil treatment and the involvement of endothelial cells transdifferentiation into smooth muscle cells in the process of hypoxia‐induced PH and PVR. Myocardin and relative markers were investigated in animal models and cultured endothelial cells. Mean pulmonary artery pressure (mPAP) was measured. Immunohistochemistry and immunofluorescence were used to show the expression of smooth muscle α‐actin (SMA), in situ hybridization (ISH) and reverse transcription polymerase chain reaction (RT‐PCR) were performed respectively to detect the myocardin and SMA expression at mRNA levels. Small interfering RNA (siRNA) induced suppression of myocardin in cultured cells. We confirmed that hypoxia induced the PH and PVR in rats. Sildenafil could attenuate the hypoxia‐induced PH. We found that myocardin mRNA expression is upregulated significantly in the hypoxic pulmonary vessels and cultured cells but downregulated in PH with Sildenafil treatment. The porcine pulmonary artery endothelial cells (PAECs) transdifferentiate into smooth muscle‐like cells in hypoxic culture while the transdifferentiation did not occur when SiRNA of myocardin was applied. Our results suggest that myocardin gene, as a marker of smooth muscle cell differentiation, was expressed in the pulmonary vessels in hypoxia‐induced PH rats, which could be downregulated by Sildenafil treatment, as well as in hypoxic cultured endothelial cells. Hypoxia induced the transdifferentiation of endothelial cells of vessels into smooth muscle‐like cells which was regulated by myocardin.

MicroRNA-206 is involved in hypoxia-induced pulmonary hypertension through targeting of the HIF-1α/Fhl-1 pathway.

Hypoxia-induced pulmonary hypertension (PH), which is characterized by vasoconstriction and subsequent structural remodeling of blood vessels, is an important event in chronic obstructive pulmonary disease patients and in people living at high altitudes. Hypoxia-inducible factor-1α (HIF-1α) and its regulator four-and-a-half LIM (Lin-11, Isl-1 and Mec-3) domain 1 (Fhl-1) have important roles in hypoxia-induced PH. MicroRNA-206 (miR-206) is critical for myogenesis and related diseases; however, the role of miR-206 in hypoxia-induced PH is unknown. miR-206 expression was evaluated in a hypoxic rat model and in cultured hypoxic pulmonary artery smooth muscle cells (PASMCs) using real-time quantitative PCR (RT-qPCR). HIF-1α and Fhl-1 expression were evaluated using RT-qPCR, western blotting, immunohistochemistry and immunofluorescence. The function of miR-206 was assessed by transfecting miR-206 mimics and inhibitors. Dual-luciferase reporter gene assays and western blotting were performed to validate the target genes of miR-206. siRNA targeted against Fhl-1 was used to investigate the effect of Fhl-1 on miR-206. Flow cytometry was used to detect the cell cycle phase distribution in each group of PASMCs. Significant downregulation of miR-206 in hypoxic lung tissue and PASMCs was identified, whereas HIF-1α and Fhl-1 were upregulated in these samples. The expression of miR-206 in the serum was different from that in the lung tissue. Transfection of pre-miR miR-206 in hypoxic conditions led to increased expression of HIF-1α and Fhl-1 rather than abolishing hypoxia-induced HIF-1α and Fhl-1, as was expected, and promoted the entry of cells into the S phase and enhanced PASMC proliferation. Fhl-1-targeted siRNA in PASMC prevented cell proliferation and led to an increased proportion of cells in the G1 phase without altering miR-206 expression. Bioinformatic analysis and dual-luciferase reporter gene assays revealed direct evidence for miR-206 targeting of HIF-1α. In conclusion, hypoxia-induced downregulation of miR-206 promotes PH by targeting the HIF-1α/Fhl-1 pathway in PASMCs. miR-206 could be a triggering factor of early stage of hypoxia-induced PH.

SDF-1α/CXCR4 axis is involved in glucose-potentiated proliferation and chemotaxis in rat vascular smooth muscle cells

Excessive proliferation of vascular smooth muscle cells (VSMCs), which migrate from the tunica media to the subendothelial region, is one of the primary lesions involved in atherogenesis in diabetes. Here, we investigated whether high glucose potentiated the proliferation and chemotaxis of VSMCs by activating SDF‐1α/CXCR4/PI‐3K/Akt signalling. The expression of SDF‐1α, CXCR4 and PCNA was up‐regulated in tunica media of thoracic aortas by streptozotocin‐induced hyperglycaemic Sprague–Dawley rats. Exposure of primary VSMCs to high glucose (25 mM) led to the up‐regulated expression of SDF‐1α and CXCR4, activated PI‐3K/Akt signalling, and consequently promoted the proliferation and chemotaxis of VSMCs. Interestingly, the administration of SDF‐1 siRNA or neutralizing antibody against SDF‐1α abolished high glucose‐induced up‐regulation of CXCR4. Moreover, pretreatment with SDF‐1α neutralizing antibody, CXCR4 specific inhibitor (AMD3100) or PI‐3K inhibitor (LY294002) attenuated the high glucose‐potentiated proliferation and chemotaxis in VSMCs. These results suggested that high glucose activated the SDF‐1α/CXCR4/PI‐3K/Akt signalling pathway in VSMCs in an autocrine manner, which enhanced the proliferation and chemotaxis of VSMCs.

Ischaemia/reperfusion induced cardiac stem cell homing to the injured myocardium by stimulating stem cell factor expression via NF-κB pathway

Ischaemia/reperfusion (I/R) is a major cause of heart failure. Recently cardiac stem cells (CSCs) were proposed as the most appropriate cell type for heart disease therapy. However, it is still unclear whether I/R can stimulate the CSCs homing to the injured myocardium. Male Sprague–Dawley rats were subjected to a 30‐min ischaemia followed by reperfusion of different intervals. RT‐PCR, western blotting and immunohistochemistry were performed to detect stem cell factor (SCF) expression at mRNA and protein levels respectively. Activation of nuclear factor‐κB (NF‐κB) was determined by electrophoretic mobility shift assay. To assess the homing of CSCs in vivo, BrdU‐labelled CSCs were injected into AV‐groove before induction of ischaemia and examined by immunofluorescent staining in the injured myocardium after I/R. From day 3 to day 6 after reperfusion, the accumulation of CSCs was significantly elevated in the injured area, which was matched with the increased SCF expression during I/R. Pretreatment of rats with NF‐κB inhibitor, N‐acetyl‐l‐cysteine (NAC) not only suppressed NF‐κB activation induced by I/R but also attenuated SCF expression. Further analysis revealed that I/R induced phosphorylation of IκBα after 15 min of reperfusion, and the raised phosphor‐IκBα returned to the basal level at 2 h of reperfusion. In simulated I/R(SI/R) in vitro, it enhanced NF‐κB activation and SCF expression in cultured neonatal rat cardiomyocytes, which was markedly inhibited by NF‐κB decoy oligodeoxynucleotide or NAC. Taken together, our results demonstrated that I/R induced CSCs homing to the injured myocardium by stimulating myocardial SCF expression via activation of NF‐κB.

Contribution of myocardin in the hypoxia-induced phenotypic switching of rat pulmonary arterial smooth muscle cells.

BACKGROUND Hypoxic exposure contributes to the phenotypic switching of smooth muscle cells (SMCs), while the mechanisms involved in this process is not yet fully elucidated. Myocardin as a co-actor of serum reaction factor plays a crucial role in differentiation of SMCs. This study was aimed to investigate the role of myocardin in hypoxia-induced phenotypic switching of rat pulmonary arterial SMCs (PASMCs). METHODS Primary PASMCs were cultured under normoxia and hypoxia (3%O(2), 48 h) respectively, and then the cell proliferation was assessed and the expression of SM22α, osteopontin (contractile and synthetic marker of SMCs, respectively), myocardin and platelet-derived growth factor-BB (PDGF-BB) were detected. After pGCSIL-GFP-shMYOCD lentviral vector was transduced to the PASMCs, the expression of myocardin and SM22α were examined. Moreover, myocardin expression in PASMCs treated with medium enriched with PDGF-BB and conditional medium (CM) from normoxia- and hypoxia-exposed PASMCs was assessed. RESULTS Exposing PASMCs to hypoxia led to an increased cell numbers and the up-regulation of proliferating cell nuclear antigen (PCNA), osteopontin and PDGF-BB; moreover, a significant down-regulation of SM22α and myocardin was identified. Further analysis revealed that knock-down of myocardin with pGCSIL-GFP-shMYOCD vector followed by a decreased SM22α in the PASMCs, and treatment of PASMCs with either exogenous PDGF-BB or hypoxic CM led to a marked decrease of myocardin. CONCLUSIONS Our findings suggest that the decrease of myocardin in PASMCs exposed to hypoxia is partly regulated by the increase of PDGF-BB, which contributes to the phenotypic switching of PASMCs in hypoxic condition.

Mucinous cystadenocarcinoma of the breast with a basal‐like immunophenotype

Mucinous cystadenocarcinoma (MCA) of the breast is extremely rare and was only recently described as a distinct variant of invasive ductal carcinoma of the breast. A case of MCA is reported in a 41‐year‐old woman. Mammographic and ultrasonographic examinations showed an irregularly shaped 10.0 × 8.0 × 5.5 cm lesion with patching calcification in the upper outer quadrant of the left breast. The gross examination revealed that the tumor has a well‐circumscribed edge with a gelatinous cut surface and hemorrhage and necrosis were also noticed in the mass. Microscopically, the mass resembled mucinous cystic neoplasm of the ovary and pancreas closely, with cystic areas lined by columnar mucinous cells and associated with abundant extracellular and intracellular mucin, which is distinctively different from mucinous carcinoma with typically nests of low grade neoplastic cells floating in the mucin pool. The tumor cells were positive for CK7, CK20 and CDX2 were negative and displayed a typical immunophenotype of basal‐like breast cancer (ER, PR, HER2 were negative, CK5/6 and EGFR were positive). Metastatic carcinoma was identified in three of 14 axillary lymph nodes. We describe here a very unusual case of breast MCA with basal‐like immunophenotype.

Contribution of CXCR4+/PDGFRβ+ progenitor cells in hypoxic alveolar arterioles muscularization: role of myocardin

AIMS Bone marrow (BM) progenitor cells may contribute to vascular remodelling. The present study aimed to investigate the contribution of BM-derived CXCR4(+) (a CXC chemokine receptor) and PDGFRbeta(+) (platelet-derived growth factor receptor beta) progenitor cells in hypoxia-induced muscularization of alveolar arterioles. METHODS AND RESULTS Accumulation of GFP(+) (green fluorescent protein) cells was markedly increased in the pulmonary vasculature by the hypoxic (10% O(2,) 4 weeks) chimeric mice with transgenic GFP-tagged BM. After injection of BM-derived CXCR4(+)/PDGFRbeta(+) progenitor cells into C57BL/6J mice, followed by 6-week hypoxia, the cells were found to home to the alveolar arterioles and readily differentiated into smooth muscle cells (SMCs). Under the same hypoxic conditions, mice infused with myocardin lentiviral RNAi vector-transduced progenitor cells displayed lower myocardin expression in the muscularized alveolar arterioles, correlating with decreased pulmonary artery pressure and arteriole muscularization. In vitro experiments further confirmed that the differentiation of the progenitor cells into SMCs occurred under hypoxia (1% O(2)), and SMC differentiation could be suppressed when myocardin RNAi was administered. CONCLUSION Theses results suggest that myocardin may contribute to the differentiation of CXCR4(+)/PDGFRbeta(+) progenitor cells into SMCs induced by hypoxia, which leads to the muscularization of alveolar arterioles.

Clinicopathological analysis of central and extraventricular neurocytoma: A report of 17 cases

SummaryNeurocytoma, a rare brain tumor, is characterized by a mass located mainly in cerebral ventricles. It is prone to be misdiagnosed as oligodendroglioma or ependymoma due to their similar histopathological features in clinical practice. This study aimed to examine the clinicopathological features and differential diagnosis of central and extraventricular neurocytoma. The clinical and histopathological data of 17 patients (male: female=7:10; age: 4–41 years; mean age: 27.4 years) with central or extraventricular neurocytoma were retrospectively analyzed. These patients showed typical radiological, histopathological and immunohistochemical features of neurocytoma. The tumor tissue was found to be composed of small uniform cells with round nuclei and clear cytoplasm resembling that of oligodendroglioma and ependymoma. Immunohistochemistry revealed the tumor tissues were positive for neuronal markers such as synaptophysin (SYN) and neuronal nuclear antigen (NeuN). It was concluded histopathological features of neurocytoma overlaps with some tumors in the central neural system. Immunopositivity for SYN and NeuN can help differentially diagnose neurocytoma.Neurocytoma, a rare brain tumor, is characterized by a mass located mainly in cerebral ventricles. It is prone to be misdiagnosed as oligodendroglioma or ependymoma due to their similar histopathological features in clinical practice. This study aimed to examine the clinicopathological features and differential diagnosis of central and extraventricular neurocytoma. The clinical and histopathological data of 17 patients (male: female=7:10; age: 4–41 years; mean age: 27.4 years) with central or extraventricular neurocytoma were retrospectively analyzed. These patients showed typical radiological, histopathological and immunohistochemical features of neurocytoma. The tumor tissue was found to be composed of small uniform cells with round nuclei and clear cytoplasm resembling that of oligodendroglioma and ependymoma. Immunohistochemistry revealed the tumor tissues were positive for neuronal markers such as synaptophysin (SYN) and neuronal nuclear antigen (NeuN). It was concluded histopathological features of neurocytoma overlaps with some tumors in the central neural system. Immunopositivity for SYN and NeuN can help differentially diagnose neurocytoma.

Composite tumor of metanephric adenoma and Wilms’ tumor of the kidney: A case report and review of the literature

Metanephric adenoma (MA) and Wilms’ tumor (WT) are two distinct types of renal tumors. Composite MA and WT of the kidney are extremely rare. Here, a rare case of composite MA and WT of the kidney in a 36-year-old male is described. MA and WT each have their own histopathological features, respectively, and they focally share morphological similarities, which can be a diagnostic challenge. Immunohistochemistry is useful in the differential diagnosis of MA and WT. The histopathological features and differential diagnosis of the composite tumor are emphasized here to promote a better and broader understanding of this less understood subject.

Astilbin inhibits proliferation of rat aortic smooth muscle cells induced by angiotensin II and down-regulates expression of protooncogene

SummaryThis study examined the effect of astilbin on the proliferation of rat aortic smooth muscle cells (RASMCs) induced by angiotensin II (AngII) and explored the possible mechanisms. Cell proliferation model of RASMCs was induced by treatmente with AngII. Cells were randomly divided to 8 groups. Normally cultured VSMCs serves as blank control group; in AngII model group, cells were treated with AngII at 10−7 mol/L; in three astilbin groups, cells were treated with 10, 15, 30 mg/L of astilbin; in three AngII+astilbin groups, cells were treated with AngII (at 10−7 mol/L) and astilbin at 10, 15, 30 mg/L. Cell proliferation ability was detected by MTT method and the cell cycles and proliferation index were flow cytometrically determined. The expression of c-myc mRNA was assessed by using reverse transcription polymerase chain reaction (RT-PCR), and the expression of NF-κB in RASMCs was immunocytochemically observed. Our results showed that MTT metabolism in RASMCs in the basic and AngII stimulated situation was inhibited by astilbin, and the cells numbers of G0/G1 phase were increased and that of G2/S phase were decreased markedly. Not only highly expression of c-myc gene stimulated by AngII could be inhibited by Astilbin significantly, but also the expression of NF-κB protein can be down regulated by Astilbin. We are led to conclude that astilbin astilbin can inhibit the AngII-mediated proliferation of RASMCs by blocking the transition of RASMCs from G0/G1 phase to S phase and by down-regulating the expression of NF-κB, c-myc gene.This study examined the effect of astilbin on the proliferation of rat aortic smooth muscle cells (RASMCs) induced by angiotensin II (AngII) and explored the possible mechanisms. Cell proliferation model of RASMCs was induced by treatmente with AngII. Cells were randomly divided to 8 groups. Normally cultured VSMCs serves as blank control group; in AngII model group, cells were treated with AngII at 10−7 mol/L; in three astilbin groups, cells were treated with 10, 15, 30 mg/L of astilbin; in three AngII+astilbin groups, cells were treated with AngII (at 10−7 mol/L) and astilbin at 10, 15, 30 mg/L. Cell proliferation ability was detected by MTT method and the cell cycles and proliferation index were flow cytometrically determined. The expression of c-myc mRNA was assessed by using reverse transcription polymerase chain reaction (RT-PCR), and the expression of NF-κB in RASMCs was immunocytochemically observed. Our results showed that MTT metabolism in RASMCs in the basic and AngII stimulated situation was inhibited by astilbin, and the cells numbers of G0/G1 phase were increased and that of G2/S phase were decreased markedly. Not only highly expression of c-myc gene stimulated by AngII could be inhibited by Astilbin significantly, but also the expression of NF-κB protein can be down regulated by Astilbin. We are led to conclude that astilbin astilbin can inhibit the AngII-mediated proliferation of RASMCs by blocking the transition of RASMCs from G0/G1 phase to S phase and by down-regulating the expression of NF-κB, c-myc gene.