Shoukang Zhu

Deficient Smad7 expression: A putative molecular defect in scleroderma

Scleroderma is a chronic systemic disease that leads to fibrosis of affected organs. Transforming growth factor (TGF) β has been implicated in the pathogenesis of scleroderma. Smad proteins are signaling transducers downstream from TGF-β receptors. Three families of Smads have been identified: (i) receptor-regulated Smad2 and -3 (R-Smads); (ii) common partner Smad4 (Co-Smad); and (iii) inhibitory Smad6 and -7 (I-Smads, part of a negative feedback loop). We have investigated the signaling components for the TGF-β pathway and TGF-β activity in scleroderma lesions in vivo and in scleroderma fibroblasts in vitro. Basal level and TGF-β-inducible expression of Smad7 are selectively decreased, whereas Smad3 expression is increased both in scleroderma skin and in explanted scleroderma fibroblasts in culture. TGF-β signaling events, including phosphorylation of Smad2 and -3, and transcription of the PAI-1 gene are increased in scleroderma fibroblasts, relative to normal fibroblasts. In vitro adenoviral gene transfer with Smad7 restores normal TGF-β signaling in scleroderma fibroblasts. These results suggest that alterations in the Smad pathway, including marked Smad7 deficiency and Smad3 up-regulation, may be responsible for TGF-β hyperresponsiveness observed in scleroderma.

Antitumorigenesis of antioxidants in a transgenic Rac1 model of Kaposi's sarcoma

Kaposis sarcoma (KS) is the major AIDS-associated malignancy. It is characterized by the proliferation of spindle cells, inflammatory infiltrate, and aberrant angiogenesis caused by Kaposis sarcoma herpesvirus (KSHV) infection. Small GTPase Rac1, an inflammatory signaling mediator triggering reactive oxygen species (ROS) production by NADPH-oxidases, is implicated in carcinogenesis and tumor angiogenesis. Here, we show that expression of a constitutively active Rac1 (RacCA) driven by the α-smooth muscle actin promoter in transgenic mice is sufficient to cause KS-like tumors through mechanisms involving ROS-driven proliferation, up-regulation of AKT signaling, and hypoxia-inducible factor 1-α–related angiogenesis. RacCA-induced tumors expressed KS phenotypic markers; displayed remarkable transcriptome overlap with KS lesions; and were, like KS, associated with male gender. The ROS scavenging agent N-acetyl-cysteine inhibited angiogenesis and completely abrogated transgenic RacCA tumor formation, indicating a causal role of ROS in tumorigenesis. Consistent with a pathogenic role in KS, immunohistochemical analysis revealed that Rac1 is overexpressed in KSHV+ spindle cells of AIDS-KS biopsies. Our results demonstrate the direct oncogenicity of Rac1 and ROS and their contribution to a KS-like malignant phenotype, further underscoring the carcinogenic potential of oxidative stress in the context of chronic infection and inflammation. They define the RacCA transgenic mouse as a model suitable for studying the role of oxidative stress in the pathogenesis and therapy of KS, with relevance to other inflammation-related malignancies. Our findings suggest host and viral genes triggering Rac1 or ROS production as key determinants of KS onset and potential KS chemopreventive or therapeutic targets.

Transcriptional Regulation of Bim by FOXO3a and Akt Mediates Scleroderma Serum–Induced Apoptosis in Endothelial Progenitor Cells

Background— Endothelial progenitor cells (EPCs) contribute to vascular regeneration/repair and thus may protect against scleroderma vasculopathy. We aimed to determine whether circulating EPCs were reduced in scleroderma, whether scleroderma sera could induce EPC apoptosis, and, if so, what the underlying apoptotic signaling pathway was. Methods and Results— Circulating EPC levels were quantified in 54 patients with scleroderma and 18 healthy control subjects by colony-forming unit assay and flow cytometry, which revealed markedly decreased EPC levels in scleroderma patients relative to healthy subjects. Substantial apoptosis was detected in EPCs after culturing in the presence of scleroderma sera compared with normal sera. Intriguingly, depletion of the IgG fraction from scleroderma sera completely abolished the apoptotic effects. Furthermore, scleroderma sera inhibited the activation/phosphorylation of Akt, which in turn suppressed the phosphorylation and degradation of forkhead transcription factor FKHRL1 (FOXO3a), resulting in the upregulation of apoptotic protein Bim. siRNA-mediated FOXO3a and Bim knockdown substantially reduced scleroderma serum–induced EPC apoptosis. Importantly, Bim expression and baseline apoptosis were increased in EPCs freshly isolated from scleroderma patients relative to that obtained from healthy subjects. Conclusion— Scleroderma serum–induced EPC apoptosis is mediated chiefly by the Akt-FOXO3a-Bim pathway, which may account, at least in part, for the decreased circulating EPC levels in scleroderma patients.

Transforming Growth Factor-β–Induced Inhibition of Myogenesis Is Mediated Through Smad Pathway and Is Modulated by Microtubule Dynamic Stability

Abstract— The expression of muscle-specific genes associated with myogenesis is controlled by several myogenic transcription factors, including myogenin and MEF2D. Transforming growth factor-&bgr; (TGF-&bgr;) has been shown to inhibit myogenesis, yet the molecular mechanisms underlying such inhibition are not known. In the present study, TGF-&bgr; was shown to inhibit myogenin and MEF2D expression and myotube formation in C2C12 myoblasts cultured in differentiation medium in a cell density–dependent manner. Transfection of C2C12 cells with Smad7, an antagonist for TGF-&bgr;/Smad signaling, restored the capacity of these cells to differentiate in the presence of TGF-&bgr; or when cultured in growth medium at low confluence, conditions that hinder muscle differentiation. Moreover, nocodazole, a microtubule-destabilizing agent, enhanced the inhibition of myogenesis exerted by TGF-&bgr;, an effect that could be restored by tubulin-polymerizing agent taxol, both of which have been shown to affect Smad-microtubule interaction and regulate TGF-&bgr;/Smad signaling. Our results indicate that TGF-&bgr; inhibits myogenesis, at least in part, via Smad pathway, and provide evidence that low-dose pharmacological agents taxol and nocodazole can be used as a means to modulate myogenesis without affecting cell survival.

Inactivation of Monocarboxylate Transporter MCT3 by DNA Methylation in Atherosclerosis

Background—Monocarboxylate transporters (MCTs) mediate lactate transport across the plasma membrane of cells. The molecular mechanisms regulating monocarboxylate transport in smooth muscle cells (SMCs) remain poorly characterized. The aim of this study was to investigate the effects of DNA methylation on MCT expression and lactate transport in SMCs in relation to atherosclerosis. Methods and Results—MCT expression was determined by real-time reverse transcription–polymerase chain reaction, Western blotting, and immunohistochemistry in SMCs isolated from human aortas and coronary arteries. Bisulfite sequencing and confocal microscopic analysis were used to study DNA methylation and lactate transport in SMCs, respectively. Downregulation of MCT3 and impaired lactate transport were detected in proliferating/synthetic SMCs, relative to the contractile phenotype. A passage number– and atherosclerotic lesion–dependent methylation pattern of MCT3 was demonstrated in the CpG island located in exon 2. Treatment of SMCs with the demethylating agent 5-aza-2′-deoxycytidine restored MCT3 expression and normalized lactate transport. Furthermore, small interfering RNA–mediated specific MCT3 knockdown substantially stimulated SMC proliferation. Conclusions—These data indicate that DNA methylation may modify monocarboxylate transport by suppressing MCT3 expression, which could be important in regulating SMC function and the development of atherosclerosis.

Paclitaxel modulates TGFβ signaling in scleroderma skin grafts in immunodeficient mice

Background Systemic sclerosis (SSc) is characterized by excessive fibrosis and obliterative vascular lesions. Abnormal TGFβ activation is implicated in the pathogenesis of SSc. Aberrant TGFβ/Smad signaling can be controlled by stabilization of microtubules with paclitaxel. Methods and Findings SSc and healthy human skin biopsies were incubated in the presence or absence of paclitaxel followed by transplantation into severe combined immunodeficient mice. TGFβ signaling, fibrosis, and neovessel formation were evaluated by quantitative RT-PCR and immunohistochemical staining. Paclitaxel markedly suppressed Smad2 and Smad3 phosphorylation and collagen deposition in SSc grafts. As a result, the autonomous maintenance/reconstitution of the SSc phenotype was prevented. Remarkably, SSc grafts showed a 2-fold increase in neovessel formation relative to normal grafts, regardless of paclitaxel treatment. Angiogenesis in SSc grafts was associated with a substantial increase in mouse PECAM-1 expression, indicating the mouse origin of the neovascular cells. Conclusion Low-dose paclitaxel can significantly suppress TGFβ/Smad activity and lessen fibrosis in SCID mice. Transplantation of SSc skin into SCID mice elicits a strong angiogenesis—an effect not affected by paclitaxel. Although prolonged chemotherapy with paclitaxel at higher doses is associated with pro-fibrotic and anti-angiogenic changes, the findings described here indicate that low-dose paclitaxel may have therapeutic benefits for SSc via modulating TGFβ signaling.

Aging in the Atherosclerosis Milieu May Accelerate the Consumption of Bone Marrow Endothelial Progenitor Cells

Objective—We have demonstrated that bone marrow cells from young and wild-type (WT), but not old apoE−/−, mice are capable of preventing atherosclerosis. This study was performed to elucidate the numerical and functional changes underlying the efficacy difference between young and old bone marrow. Methods and Results—CD34+/VEGFR2+ conventional endothelial progenitor cells and lin−/cKit+/Sca-1+ hematopoietic stem cells did not differ numerically or functionally between young and old apoE−/− bone marrow. Fluorescence- activated cell sorter analysis, however, showed that a group of cells (simple little cells or SLCs), characteristically located in the lower left quadrant of forward scatter/side scatter flow cytometric plot, were markedly decreased in old WT and apoE−/− marrow, but abundantly present in young WT and apoE−/− bone marrow. The SLC fraction was mainly composed of lin−/cKit−/Sca-1− cells. In vitro differentiation assay demonstrated substantially more efficient endothelial differentiation of lin−/cKit−/Sca-1− SLCs than other bone marrow fractions at a single cell level and en masse. Furthermore, old lin−/cKit−/Sca-1− SLCs had a trend of decreased endothelial differentiation capability. Conclusions—Lin−/cKit−/Sca-1− SLCs may represent a previously unrecognized cell population, enriched for endothelial progenitors. The identification of these cells may help improve the efficacy of cell therapy.

Upregulation of PAI-1 is mediated through TGF β /SMAD pathway in transplant arteriopathy

BACKGROUND Plasminogen activator inhibitor type 1 (PAI-1) is the primary physiologic inhibitor of plasminogen activator in vivo. Increased PAI-1 expression is associated with arteriosclerosis. Transforming growth factor-beta (TGF-beta) induces PAI-1 production via Smads. METHODS In vivo, TGF-beta receptors (TbetaRs), Smad2, Smad3, and Smad4, PAI-1, and Smad2 phosphorylation were examined by immunohistochemistry in 3 native aortas, 14 rat aortic syngrafts, and 19 allografts collected at 15, 30, and 45 days post-transplantation. In vitro, phosphorylation of Smad2 and induction of PAI-1 mRNA in human aortic smooth muscle cells (SMCs) in response to TGF-beta treatment were detected by Western blot and by TaqMan real-time RT-PCR, respectively. RESULTS Immunohistochemical staining revealed that vascular parenchymal cells contained TbetaRI, TbetaRII, Smad2, Smad3, and Smad4, known signaling transducers for TGF-beta/Smad pathway, in all samples. Intense staining for phospho-Smad2 was observed in 94% of endothelial cells (ECs), 86% of intimal cells, 27% of medial SMCs, and 38% of adventitial cells at all 3 time points in all aortic allografts, but only in 5% of ECs in syngrafts. PAI-1 immunoreactivity was detected in similar number of cells, and from consecutive sections, phospho-Smad2 colocalized with PAI-1, in the aortic allografts. Low basal level PAI-1 expression was observed in aortic syngrafts and native vessels. Smad2 phosphorylation and time-dependent PAI-1 induction were detected in cultured SMCs upon TGF-beta treatment. CONCLUSIONS Phospho-Smad2 staining in aortic allografts indicates the activation of TGF-beta signaling in allo-transplantation; and co-localization of PAI-1 and phospho-Smad2 suggests that PAI-1 upregulation is mediated mainly by TGF-beta/Smad pathway in aortic allografts.

MicroRNA-146a Induces Lineage-Negative Bone Marrow Cell Apoptosis and Senescence by Targeting Polo-Like Kinase 2 Expression.

Objective— Lineage-negative bone marrow cells (lin− BMCs) are enriched in endothelial progenitor cells and mediate vascular repair. Aging-associated senescence and apoptosis result in reduced number and functionality of lin− BMCs, impairing their prorepair capacity. The molecular mechanisms underlying lin− BMC senescence and apoptosis are poorly understood. MicroRNAs (miRNAs) regulate many important biological processes. The identification of miRNA-mRNA networks that modulate the health and functionality of lin− BMCs is a critical step in understanding the process of vascular repair. The aim of this study was to characterize the role of the miR-146a–Polo-like kinase 2 (Plk2) network in regulating lin− BMC senescence, apoptosis, and their angiogenic capability. Approach and Results— Transcriptome analysis in lin− BMCs isolated from young and aged wild-type and ApoE−/− (apolipoprotein E) mice showed a significant age-associated increase in miR-146a expression. In silico analysis, expression study and Luciferase reporter assay established Plk2 as a direct target of miR-146a. miR-146a overexpression in young lin− BMCs inhibited Plk2 expression, resulting in increased senescence and apoptosis, via p16Ink4a/p19Arf and p53, respectively, as well as impaired angiogenic capacity in vitro and in vivo. Conversely, suppression of miR-146a in aged lin− BMCs increased Plk2 expression and rejuvenated lin− BMCs, resulting in decreased senescence and apoptosis, leading to improved angiogenesis. Conclusions— (1) miR-146a regulates lin− BMC senescence and apoptosis by suppressing Plk2 expression that, in turn, activates p16Ink4a/p19Arf and p53 and (2) modulation of miR-146a or its target Plk2 may represent a potential therapeutic intervention to improve lin− BMC–mediated angiogenesis and vascular repair.

Erratum: Deficient Smad7 expression: A putative molecular defect in scleroderma (PNAS (2016) 113:15 (E2208))

MEDICAL SCIENCES Retraction for “Deficient Smad7 expression: A putative molecular defect in scleroderma,” by Chunming Dong, Shoukang Zhu, Tao Wang, Woohyun Yoon, Zhiru Li, Rene J. Alvarez, Peter ten Dijke, Barbara White, Fredrick M. Wigley, and Pascal J. GoldschmidtClermont, which appeared in issue 6, March 19, 2002, of Proc Natl Acad Sci USA (99:3908–3913; 10.1073/pnas.062010399). The authors wish to note the following: “It has recently been brought to our attention that some of the elements in Fig. 3 of our paper may have been fabricated. Unfortunately, because of the time elapsed since publication, we no longer have in our possession the original gels and blots that were used to produce the figure. While we trust that the other data in the paper is genuine and the overall conclusions sound, we have no alternative but to request a retraction of our paper. We apologize for any inconvenience this may cause.”