Xiao-Bing Cui

Response gene to complement 32 deficiency causes impaired placental angiogenesis in mice

AIMS The objectives of this study are to determine the role of response gene to complement 32 (RGC-32) in the placental angiogenesis during pregnancy and explore the underlying mechanisms. METHODS AND RESULTS RGC-32-deficient (RGC32(-/-)) mice were generated from C57BL/6 embryonic stem cells with deletion of exon 2 and 3 of the RGC-32 gene. Most of the RGC32(-/-) mice can survive. However, their body sizes were much smaller compared with their wild-type littermates when they were born. By examining the embryo development and placentas at 16.5 days post-coitum, we found that RGC32(-/-) embryos and foetal placentas were significantly smaller than the wild-type. Further analysis showed that the labyrinth zone of RGC32(-/-) placenta was smaller with defective angiogenesis. Mechanistically, vascular endothelial growth factor (VEGF) receptor 2 (VEGFR2) and placental growth factor (PlGF) were significantly down-regulated in RGC32(-/-) placentas, suggesting that VEGFR2 and PlGF may mediate RGC-32 function in placental angiogenesis. Indeed, knockdown of RGC-32 by shRNA inhibited VEGF-induced endothelial cell proliferation, migration, and tube formation while blocking VEGFR2 expression. RGC-32 appeared to regulate VEGFR2 expression via activation of NF-kB. Moreover, RGC-32 regulated trophoblasts proliferation via control of PlGF expression. CONCLUSION Absence of RGC-32 caused foetal growth restriction through interrupting the placental angiogenesis, which was due to the decrease in VEGFR2 expression through the NF-kB-dependent pathway in endothelial cells and PlGF expression in trophoblasts.

ADAR1-Mediated RNA Editing, A Novel Mechanism Controlling Phenotypic Modulation of Vascular Smooth Muscle Cells

RATIONALE Vascular smooth muscle cell (SMC) phenotypic modulation is characterized by the downregulation of SMC contractile genes. Platelet-derived growth factor-BB, a well-known stimulator of SMC phenotypic modulation, downregulates SMC genes via posttranscriptional regulation. The underlying mechanisms, however, remain largely unknown. OBJECTIVE To establish RNA editing as a novel mechanism controlling SMC phenotypic modulation. METHODS AND RESULTS Precursor mRNAs (pre-mRNA) of SMC myosin heavy chain and smooth muscle α-actin were accumulated while their mature mRNAs were downregulated during SMC phenotypic modulation, suggesting an abnormal splicing of the pre-mRNAs. The abnormal splicing resulted from SMC marker pre-mRNA editing that was facilitated by adenosine deaminase acting on RNA 1 (ADAR1), an enzyme converting adenosines to inosines (A→I editing) in RNA sequences. ADAR1 expression inversely correlated with SMC myosin heavy chain and smooth muscle α-actin levels; knockdown of ADAR1 restored SMC myosin heavy chain and smooth muscle α-actin expression in phenotypically modulated SMC, and editase domain mutation diminished the ADAR1-mediated abnormal splicing of SMC marker pre-mRNAs. Moreover, the abnormal splicing/editing of SMC myosin heavy chain and smooth muscle α-actin pre-mRNAs occurred during injury-induced vascular remodeling. Importantly, heterozygous knockout of ADAR1 dramatically inhibited injury-induced neointima formation and restored SMC marker expression, demonstrating a critical role of ADAR1 in SMC phenotypic modulation and vascular remodeling in vivo. CONCLUSIONS Our results unraveled a novel molecular mechanism, that is, pre-mRNA editing, governing SMC phenotypic modulation.

RGC-32 Deficiency Protects against Hepatic Steatosis by Reducing Lipogenesis

Background: Hepatic steatosis is an increasing health concern associated with metabolic syndrome. Results: RGC-32 deficiency protects mice from HFD- and ethanol-induced hepatic steatosis by decreasing hepatic lipogenic gene expression. Conclusion: RGC-32 is a novel regulator for hepatic lipogenesis. Significance: RGC-32 may be a novel target for treating hepatic steatosis. Hepatic steatosis is associated with insulin resistance and metabolic syndrome because of increased hepatic triglyceride content. We have reported previously that deficiency of response gene to complement 32 (RGC-32) prevents high-fat diet (HFD)-induced obesity and insulin resistance in mice. This study was conducted to determine the role of RGC-32 in the regulation of hepatic steatosis. We observed that hepatic RGC-32 was induced dramatically by both HFD challenge and ethanol administration. RGC-32 knockout (RGC32−/−) mice were resistant to HFD- and ethanol-induced hepatic steatosis. The hepatic triglyceride content of RGC32−/− mice was decreased significantly compared with WT controls even under normal chow conditions. Moreover, RGC-32 deficiency decreased the expression of lipogenesis-related genes, sterol regulatory element binding protein 1c (SREBP-1c), fatty acid synthase, and stearoyl-CoA desaturase 1 (SCD1). RGC-32 deficiency also decreased SCD1 activity, as indicated by decreased desaturase indices of the liver and serum. Mechanistically, insulin and ethanol induced RGC-32 expression through the NF-κB signaling pathway, which, in turn, increased SCD1 expression in a SREBP-1c-dependent manner. RGC-32 also promoted SREBP-1c expression through activating liver X receptor. These results demonstrate that RGC-32 contributes to the development of hepatic steatosis by facilitating de novo lipogenesis through activating liver X receptor, leading to the induction of SREBP-1c and its target genes. Therefore, RGC-32 may be a potential novel drug target for the treatment of hepatic steatosis and its related diseases.

CTP Synthase 1, a Smooth Muscle–Sensitive Therapeutic Target for Effective Vascular Repair

Objective—Vascular remodeling as a result of smooth muscle cell (SMC) proliferation and neointima formation is a major medical challenge in cardiovascular intervention. However, antineointima drugs often indistinguishably block re-endothelialization, an essential step toward successful vascular repair, because of their nonspecific effect on endothelial cells (ECs). The objective of this study is to identify a therapeutic target that differentially regulates SMC and EC proliferation. Approach and Results—Using both rat balloon injury and mouse wire injury models, we identified CTP synthase 1 (CTPS1) as one of the potential targets that may be used for developing therapeutics for treating neointima-related disorders. CTPS1 was induced in proliferative SMCs in vitro and neointima SMCs in vivo. Blockade of CTPS1 expression by small hairpin RNA or activity by cyclopentenyl cytosine suppressed SMC proliferation and neointima formation. Surprisingly, cyclopentenyl cytosine had much less effect on EC proliferation. Of importance, blockade of CTPS1 in vivo sustained the re-endothelialization as a result of induction of CTP synthesis salvage pathway enzymes nucleoside-diphosphate kinase A and B in ECs. Diphosphate kinase B seemed to preserve EC proliferation via use of extracellular cytidine to synthesize CTP. Indeed, blockade of both CTPS1 and diphosphate kinase B suppressed EC proliferation in vitro and the re-endothelialization in vivo. Conclusions—Our study uncovered a fundamental difference in CTP biosynthesis between SMCs and ECs during vascular remodeling, which provided a novel strategy by using cyclopentenyl cytosine or other CTPS1 inhibitors to selectively block SMC proliferation without disturbing or even promoting re-endothelialization for effective vascular repair after injury.

RGC-32 (Response Gene to Complement 32 Protein) Deficiency Protects Endothelial Cells From Inflammation and Attenuates Atherosclerosis

Objective— The objective of this study is to determine the role and underlying mechanisms of RGC-32 (response gene to complement 32 protein) in atherogenesis. Approach and Results— RGC-32 was mainly expressed in endothelial cells of atherosclerotic lesions in both ApoE−/− (apolipoprotein E deficient) mice and human patients. Rgc-32 deficiency (Rgc32−/−) attenuated the high-fat diet–induced and spontaneously developed atherosclerotic lesions in ApoE−/− mice without affecting serum cholesterol concentration. Rgc32−/− seemed to decrease the macrophage content without altering collagen and smooth muscle contents or lesional macrophage proliferation in the lesions. Transplantation of WT (wild type) mouse bone marrow to lethally irradiated Rgc32−/− mice did not alter Rgc32−/−-caused reduction of lesion formation and macrophage accumulation, suggesting that RGC-32 in resident vascular cells, but not the macrophages, plays a critical role in the atherogenesis. Of importance, Rgc32−/− decreased the expression of ICAM-1 (intercellular adhesion molecule-1) and VCAM-1 (vascular cell adhesion molecule-1) in endothelial cells both in vivo and in vitro, resulting in a decrease in TNF-&agr; (tumor necrosis factor-&agr;)–induced monocyte–endothelial cell interaction. Mechanistically, RGC-32 mediated the ICAM-1 and VCAM-1 expression, at least partially, through NF (nuclear factor)-&kgr;B signaling pathway. RGC-32 directly interacted with NF-&kgr;B and facilitated its nuclear translocation and enhanced TNF-&agr;–induced NF-&kgr;B binding to ICAM-1 and VCAM-1 promoters. Conclusions— RGC-32 mediates atherogenesis by facilitating monocyte–endothelial cell interaction via the induction of endothelial ICAM-1 and VCAM-1 expression, at least partially, through NF-&kgr;B signaling pathway.

Olfactomedin 2 Regulates Smooth Muscle Phenotypic Modulation and Vascular Remodeling Through Mediating Runt-Related Transcription Factor 2 Binding to Serum Response Factor.

Objective— The objective of this study is to investigate the role and underlying mechanism of Olfactomedin 2 (Olfm2) in smooth muscle cell (SMC) phenotypic modulation and vascular remodeling. Approach and Results— Platelet-derived growth factor-BB induces Olfm2 expression in primary SMCs while modulating SMC phenotype as shown by the downregulation of SMC marker proteins. Knockdown of Olfm2 blocks platelet-derived growth factor-BB–induced SMC phenotypic modulation, proliferation, and migration. Conversely, Olfm2 overexpression inhibits SMC marker expression. Mechanistically, Olfm2 promotes the interaction of serum response factor with the runt-related transcription factor 2 that is induced by platelet-derived growth factor-BB, leading to a decreased interaction between serum response factor and myocardin, causing a repression of SMC marker gene transcription and consequently SMC phenotypic modulation. Animal studies show that Olfm2 is upregulated in balloon-injured rat carotid arteries. Knockdown of Olfm2 effectively inhibits balloon injury–induced neointima formation. Importantly, knockout of Olfm2 in mice profoundly suppresses wire injury–induced neointimal hyperplasia while restoring SMC contractile protein expression, suggesting that Olfm2 plays a critical role in SMC phenotypic modulation in vivo. Conclusions— Olfm2 is a novel factor mediating SMC phenotypic modulation. Thus, Olfm2 may be a potential target for treating injury-induced proliferative vascular diseases.

Response by Cui et al to Letter Regarding Article, “RGC-32 (Response Gene to Complement 32) Deficiency Protects Endothelial Cells From Inflammation and Attenuates Atherosclerosis”

We welcome the letter from Rus et al commenting on our recent study showing that RGC-32 (response gene to complement 32) mediates the development of atherosclerosis by facilitating monocyte–endothelial cell (EC) interaction through induction of endothelial intercellular adhesion molecule-1 and vascular cell adhesion molecule-1.1 Our conclusion is based on several observations: (1) RGC-32 is induced in ECs in both human and mouse atherosclerotic lesions; (2) RGC-32 deficiency ( Rgc32 −/−) attenuates the spontaneously-developed and high-fat diet–induced atherosclerosis in Apoe −/− mice; (3) Rgc32 −/− mice transplanted with wild-type bone marrow does not significantly alter the Rgc32 −/− phenotype; (4) Rgc32 −/− inhibits endothelial intercellular adhesion molecule-1 and vascular cell adhesion molecule-1expression and monocyte–EC interaction.1 These data strongly suggest that endothelial RGC-32 plays an essential role in atherosclerosis. We understand the concern in the letter about the differences in RGC-32 expression in human atherosclerotic lesions in our and Dr Vlaicu et al’s2 studies and appreciate the insights that the discrepancy may be the result of the differences in the atherosclerotic lesions from different arterial regions. …

Janus Kinase 3, a Novel Regulator for Smooth Muscle Proliferation and Vascular Remodeling

Objective— Vascular remodeling because of smooth muscle cell (SMC) proliferation is a common process occurring in several vascular diseases, such as atherosclerosis, aortic aneurysm, post-transplant vasculopathy, restenosis after angioplasty, etc. The molecular mechanism underlying SMC proliferation, however, is not completely understood. The objective of this study is to determine the role and mechanism of Janus kinase 3 (JAK3) in vascular remodeling and SMC proliferation. Approach and Results— Platelet-derived growth factor-BB, an SMC mitogen, induces JAK3 expression and phosphorylation while stimulating SMC proliferation. Janex-1, a specific inhibitor of JAK3, or knockdown of JAK3 by short hairpin RNA, inhibits the SMC proliferation. Conversely, ectopic expression of JAK3 promotes SMC proliferation. Mechanistically, JAK3 promotes the phosphorylation of signal transducer and activator of transcription 3 and c-Jun N-terminal kinase in SMC, 2 signaling pathways known to be critical for SMC proliferation and vascular remodeling. Blockade of these 2 signaling pathways by their inhibitors impeded the JAK3-mediated SMC proliferation. In vivo, knockdown of JAK3 attenuates injury-induced neointima formation with attenuated neointimal SMC proliferation. Knockdown of JAK3 also induces neointimal SMC apoptosis in rat carotid artery balloon injury model. Conclusions— Our results demonstrate that JAK3 mediates SMC proliferation and survival during injury-induced vascular remodeling, which provides a potential therapeutic target for preventing neointimal hyperplasia in proliferative vascular diseases.