Anlin Liang

The Mechanical Stress–Activated Serum-, Glucocorticoid-Regulated Kinase 1 Contributes to Neointima Formation in Vein Grafts

Rationale: Mechanical stress plays an important role in proliferation of venous smooth muscle cells (SMCs) in neointima, a process of formation that contributes to failure of vein grafts. However, it is unknown what intracellular growth signal leads to proliferation of venous SMCs. Objective: The objective of this study is to identify mechanisms of mechanical stretch on neointima formation. Methods and Results: By a microarray analysis, we found that mechanical cyclic stretch (15% elongation) stimulated the transcription of SGK-1 (serum-, glucocorticoid-regulated kinase-1). Mechanical stretch–induced SGK-1 mRNA expression was blocked by actinomycin D. The mechanism for the SGK-1 expression involved MEK1 but not p38 or JNK signaling pathway. SGK-1 activation in response to stretch is blocked by insulin-like growth factor (IGF)-1 receptor inhibitor and mammalian target of rapamycin complex (mTORC)2 inhibitor (Ku-0063794) but not mTORC1 inhibitor (rapamycin). Mechanical stretch–induced bromodeoxyuridine incorporation was reduced by 83.5% in venous SMCs isolated from SGK-1 knockout mice. In contrast, inhibition of Akt, another downstream signal of PI3K resulted in only partial inhibition of mechanical stretch–induced proliferation of venous SMCs. Mechanical stretch also induced phosphorylation and nuclear exportation of p27kip1, whereas knockout of SGK-1 attenuated this effect of mechanical stretch on p27kip1. In vivo, we found that placement of a vein graft into artery increased SGK-1 expression. Knockout of SGK-1 effectively prevented neointima formation in vein graft. There is significant lower level of p27kip1 located in the nucleus of neointima cells in SGK-1 knockout mice compared with that of wild-type vein graft. In addition, we also found that wire injury of artery or growth factors in vitro increased expression of SGK-1. Conclusions: These results suggest that SGK-1 is an injury-responsive kinase that could mediate mechanical stretch–induced proliferation of vascular cells in vein graft, leading to neointima formation.

FSP-1 Silencing in Bone Marrow Cells Suppresses Neointima Formation in Vein Graft

Rationale: Fibroblast-specific protein 1 (FSP-1) plays multiple roles in promoting cell proliferation and motility. Increased FSP-1 expression in smooth muscle cells (SMCs) has been associated with their enhanced proliferation. Objective: To study how FSP-1 contributes to neointima formation of vein grafts. Methods: Arteriovenous grafts were created in wild-type or FSP-1–GFP mice (green fluorescent protein expression regulated by FSP-1 promoter). The effects of FSP-1 on bone marrow (BM) cell migration and on SMC proliferation were studied in vivo and in vitro. Results: On creation of a vein graft, there was rapid deposition of platelets on the denuded surface leading to secretion of the chemokine stromal cell–derived factor-1&agr; (SDF-1&agr;). This was followed by recruitment of BM-derived cells expressing the SDF-1&agr; receptor CXCR4; homing of FSP-1–positive cells was found to be dependent on platelet-derived SDF-1&agr;. FSP-1 was expressed in 8% of the BM cells, and 20% of these express CD45; 85% of FSP-1–positive cells express CD11b. We found that the FSP-1–positive cells migrated into the vein graft in a Rac-1–dependent fashion. FSP-1 expression was also found to stimulate proliferation of SMCs through a MEK5-ERK5 signaling pathway that can be suppressed by a dominant-negative Rac1. Consequently, knocking down FSP-1 expression in BM cells prevented neointimal formation. Conclusions: BM-derived FSP-1+ cells enhance neointima formation through an increase in transendothelial invasion with stimulation of SMC proliferation. The Rac1 and ERK5 signaling cascade mediate FSP-1–induced responses in SMCs and BM cells. This novel pathophysiology suggests a new therapeutic target, FSP-1, for preventing the development of neointima in vein grafts.

Blocking Notch in Endothelial Cells Prevents Arteriovenous Fistula Failure Despite CKD

Neointima formation causes the failure of 60% of arteriovenous fistulas (AVFs) within 2 years. Neointima-forming mechanisms are controversial but possibly linked to excess proinflammatory responses and dysregulated Notch signaling. To identify how AVFs fail, we anastomosed the carotid artery to the internal jugular vein in normal and uremic mice and compared these findings with those in failed AVFs from patients with ESRD. Endothelial cells (ECs) of AVFs in uremic mice or patients expressed mesenchymal markers (FSP-1 and/or α-SMA) and exhibited increased expression and nuclear localization of Notch intracellular domain compared with ECs of AVFs in pair-fed control mice. Furthermore, expression of VE-Cadherin decreased, whereas expression of Notch1 and -4, Notch ligands, the downstream transcription factor of Notch, RBP-Jκ, and Notch target genes increased in ECs of AVFs in uremic mice. In cultured ECs, ectopic expression of Notch ligand or treatment with TGF-β1 triggered the expression of mesenchymal markers and induced endothelial cell barrier dysfunction, both of which were blocked by Notch inhibition or RBP-Jκ knockout. Furthermore, Notch-induced defects in barrier function, invasion of inflammatory cells, and neointima formation were suppressed in mice with heterozygous knockdown of endothelial-specific RBP-Jκ. These results suggest that increased TGF-β1, a complication of uremia, activates Notch in endothelial cells of AVFs, leading to accelerated neointima formation and AVF failure. Suppression of Notch activation could be a strategy for improving AFV function in uremia.

Protective Role of Insulin-Like Growth Factor-1 Receptor in Endothelial Cells against Unilateral Ureteral Obstruction–Induced Renal Fibrosis

Insulin-like growth factor-1 receptor (IGF-1R) can regulate vascular homeostasis and endothelial function. We studied the role of IGF-1R in oxidative stress-induced endothelial dysfunction. Unilateral ureteral obstruction (UUO) was performed in wild-type (WT) mice and mice with endothelial cell (EC)-specific IGF-1R knockout (KO). After UUO in endothelial IGF-1R KO mice, endothelial barrier dysfunction was more severe than in WT mice, as seen by increased inflammatory cell infiltration and vascular endothelial (VE)-cadherin phosphorylation. UUO in endothelial IGF-1R KO mice increased interstitial fibroblast accumulation and enhanced extracellular protein deposition as compared with the WT mice. Endothelial barrier function measured by transendothelial migration in response to hydrogen peroxide (H2O2) was impaired in ECs. Silencing IGF-1R enhanced the influence of H2O2 in disrupting the VE-protein tyrosine phosphatase/VE-cadherin interaction. Overexpression of IGF-1R suppressed H2O2-induced endothelial barrier dysfunction. Furthermore, by using the piggyBac transposon system, we expressed IGF-1R in VE cells in mice. The expression of IGF-1R in ECs also suppressed the inflammatory cell infiltration and renal fibrosis induced by UUO. IGF-1R KO in the VE-cadherin lineage of bone marrow cells had no significant effect on the UUO-induced fibrosis, as compared with control mice. Our results indicate that IGF-1R in the endothelium maintains the endothelial barrier function by stabilization of the VE-protein tyrosine phosphatase/VE-cadherin complex. Decreased expression of IGF-1R impairs endothelial function and increases the fibrosis of kidney disease.

Loss of glutathione S-transferase A4 accelerates obstruction-induced tubule damage and renal fibrosis

Glutathione transferase isozyme A4 (GSTA4) exhibits high catalytic efficiency to metabolize 4‐hydroxynonenal (4‐HNE), a highly reactive lipid peroxidation product that has been implicated in the pathogenesis of various chronic diseases. We investigated the role of 4‐HNE in the mechanisms of unilateral ureteral obstruction (UUO)‐induced fibrosis and its modulation by GSTA4‐4 in a mouse model. Our data indicate that after UUO, accumulation of 4‐HNE and its adducts were increased in renal tissues, with a concomitant decrease in the expression of GSTA4‐4 in mice. As compared to wild‐type (WT) mice, UUO caused an increased expression of fibroblast markers in the interstitium of GSTA4 KO mice. Additionally, increased autophagy and tubular cell damage were more severe in UUO‐treated GSTA4 KO mice than in WT mice. Furthermore, GSK‐3β phosphorylation and expression of Snail, a regulator of E‐cadherin and Occludin, was found to be significantly higher in UUO‐inflicted GSTA4 KO mice. GSTA4 over‐expression prevented 4‐HNE‐induced autophagy activation, tubular cell damage and Snail nuclear translocation in vitro. The effects of long‐term expression of GSTA4 in restoration of UUO‐induced damage in mice with the GSTA4 inducible transposon system indicated that release of obstruction after 3 days of UUO resulted in the attenuation of interstitial SMAα and collagen I expression. This transposon‐delivered GSTA4 expression also suppressed UUO‐induced loss of tubular cell junction markers and autophagy activation. Together, these results indicate that 4‐HNE significantly contributes to the mechanisms of tubule injury and fibrosis and that these effects can be inhibited by the enhanced expression of GSTA4‐4. Copyright

Migration of smooth muscle cells from the arterial anastomosis of arteriovenous fistulas requires Notch activation to form neointima.

A major factor contributing to failure of arteriovenous fistulas (AVFs) is migration of smooth muscle cells into the forming neointima. To identify the source of smooth muscle cells in neointima, we created end-to-end AVFs by anastomosing the common carotid artery to the jugular vein and studied neural crest-derived smooth muscle cells from the carotid artery which are Wnt1-positive during development. In Wnt1-cre-GFP mice, smooth muscle cells in the carotid artery but not the jugular vein are labeled with GFP. About half of the cells were GFP-positive in the neointima indicating their migration from the carotid artery to the jugular vein in AVFs created in these mice. Since fibroblast-specific protein-1 (FSP-1) regulates smooth muscle cell migration, we examined FSP-1 in failed AVFs and polytetrafluoroethylene (PTFE) grafts from patients with ESRD or from AVFs in mice with chronic kidney disease. In smooth muscle cells of AVFs or PTFE grafts, FSP-1 and activation of Notch1 are present. In smooth muscle cells, Notch1 increased RBP-Jκ transcription factor activity and RBP-Jκ stimulated FSP-1 expression. Conditional knockout of RBP-Jκ in smooth muscle cells or general knockout of FSP-1, suppressed neointima formation in AVFs in mice. Thus, the artery of AVFs is the major source of smooth muscle cells during neointima formation. Knockout of RBP-Jκ or FSP-1 ameliorates neointima formation and might improve AVF patency during long-term follow up.

Serum Glucocorticoid–Regulated Kinase 1 Blocks CKD–Induced Muscle Wasting Via Inactivation of FoxO3a and Smad2/3

Muscle proteolysis in CKD is stimulated when the ubiquitin-proteasome system is activated. Serum glucocorticoid-regulated kinase 1 (SGK-1) is involved in skeletal muscle homeostasis, but the role of this protein in CKD-induced muscle wasting is unknown. We found that, compared with muscles from healthy controls, muscles from patients and mice with CKD express low levels of SGK-1. In mice, SGK-1-knockout (SGK-1-KO) induced muscle loss that correlated with increased expression of ubiquitin E3 ligases known to facilitate protein degradation by the ubiquitin-proteasome, and CKD substantially aggravated this response. SGK-1-KO also altered the phosphorylation levels of transcription factors FoxO3a and Smad2/3. In C2C12 muscle cells, expression of dominant negative FoxO3a or knockdown of Smad2/3 suppressed the upregulation of E3 ligases induced by loss of SGK-1. Additionally, SGK-1 overexpression increased the level of phosphorylated N-myc downstream-regulated gene 1 protein, which directly interacted with and suppressed the phosphorylation of Smad2/3. Overexpression of SGK-1 in wild-type mice with CKD had similar effects on the phosphorylation of FoxO3a and Smad2/3 and prevented CKD-induced muscle atrophy. Finally, mechanical stretch of C2C12 muscle cells or treadmill running of wild-type mice with CKD stimulated SGK-1 production, and treadmill running inhibited proteolysis in muscle. These protective responses were absent in SGK-1-KO mice. Thus, SGK-1 could be a mechanical sensor that mediates exercise-induced improvement in muscle wasting stimulated by CKD.

Impaired Integrin β3 Delays Endothelial Cell Regeneration and Contributes to Arteriovenous Graft Failure in Mice

Objective— Neointima formation is associated with stenosis and subsequent thrombosis in arteriovenous grafts (AVGs). A role of integrin &bgr;3 in the neointima formation of AVGs remains poorly understood. Approach and Results— In integrin &bgr;3−/− mice, we found significantly accelerated occlusion of AVGs compared with the wild-type mice. This is caused by the development of neointima and lack of endothelial regeneration. The latter is a direct consequence of impaired functions of circulating angiogenic cells (CACs) and platelets in integrin &bgr;3−/− mice. Evidence suggests the involvement of platelet regulating CAC homing to and differentiation at graft sites via transforming growth factor-&bgr;1 and Notch signaling pathway. First, CACs deficient of integrin &bgr;3 impaired adhesion activity toward exposed subendothelium. Second, platelets from integrin &bgr;3−/− mice failed to sufficiently stimulate CACs to differentiate into mature endothelial cells. Finally, we found that transforming growth factor-&bgr;1 level was increased in platelets from integrin &bgr;3−/− mice and resulted in enhanced Notch1 activation in CACs in AVGs. These results demonstrate that integrin &bgr;3 is critical for endothelial cell homing and differentiation. The increased transforming growth factor-&bgr;1 and Notch1 signaling mediates integrin &bgr;3−/−-induced AVG occlusion. This accelerated occlusion of AVGs was reversed in integrin &bgr;3−/− mice transplanted with the bone marrow from wild-type mice. Conclusions— Our results suggest that boosting integrin &bgr;3 function in the endothelial cells and platelets could prevent neointima and thrombosis in AVGs.

Integrin β3 Mediates the Endothelial-to-Mesenchymal Transition via the Notch Pathway

Background/Aims: Neointimal hyperplasia is responsible for stenosis, which requires corrective vascular surgery, and is also a major morphological feature of many cardiovascular diseases. This hyperplasia involves the endothelial-to-mesenchymal transition (EndMT). We investigated whether integrin β3 can modulate the EndMT, as well as its underlying mechanism. Methods: Integrin β3 was overexpressed or knocked down in human umbilical vein endothelial cells (HUVECs). The expression of endothelial markers and mesenchymal markers was determined by real-time reverse transcription PCR (RT-PCR), immunofluorescence staining, and western blot analysis. Notch signaling pathway components were detected by real-time RT-PCR and western blot analysis. Cell mobility was evaluated by wound-healing, Transwell, and spreading assays. Fibroblast-specific protein 1 (FSP-1) promoter activity was determined by luciferase assay. Results: Transforming growth factor (TGF)-β1 treatment or integrin β3 overexpression significantly promoted the EndMT by downregulating VE-cadherin and CD31 and upregulating smooth muscle actin α and FSP-1 in HUVECs, and by enhancing cell migration. Knockdown of integrin β3 reversed these effects. Notch signaling was activated after TGF-β1 treatment of HUVECs. Knockdown of integrin β3 suppressed TGF-β1-induced Notch activation and expression of the Notch downstream target FSP-1. Conclusion: Integrin β3 may promote the EndMT in HUVECs through activation of the Notch signaling pathway.