Wnt4/ß-catenin signaling induces VSMC proliferation and is associated with intimal thickening.

RATIONALE: Vascular smooth muscle cell (VSMC) proliferation causes intimal thickening in atherosclerosis and restenosis. Previously, we demonstrated that Wnt/ß-catenin signaling upregulates VSMC proliferation in vitro. OBJECTIVE: We examined this pathway in vivo and investigated the involvement of specific Wnt proteins in VSMC proliferation. METHODS AND RESULTS: Left carotid arteries of TOPgal (ß-catenin signaling reporter) transgenic mice were ligated to induce intimal thickening. ß-Catenin signaling was induced in the media and intima at 3 and 28 days after ligation, respectively, and was associated with VSMC proliferation and cyclin D1 expression. In vitro, a Wnt agonist promoted mouse VSMC proliferation, whereas Wnt inhibitory factor (WIF)-1 retarded platelet-derived growth factor-BB (PDGF-BB)-induced VSMC proliferation. Microarray analysis and quantitative PCR detected a significant induction of Wnt2 and Wnt4 mRNA in PDGF-BB-treated (proliferating) VSMCs compared to quiescent VSMCs. Western blotting revealed this increase was only translated into protein for Wnt4. Specific silencing RNA knockdown of Wnt4, but not Wnt2, significantly reduced VSMC proliferation. Recombinant Wnt4, but not Wnt2, significantly increased VSMC proliferation by ≈2-fold and silencing RNA knockdown revealed this is via Frizzled 1. Immunohistochemistry showed that increased Wnt4 protein correlated with VSMC proliferation and cyclin D1 expression (P<0.05 and P<0.001, respectively) during intimal thickening after rat carotid artery injury. Importantly, we also showed that intimal thickening and VSMC proliferation after carotid artery ligation was significantly retarded in Wnt4(+/-) compared to Wnt4(+/+) mice. CONCLUSIONS: This study demonstrates that Wnt/ß-catenin signaling occurs in proliferating VSMCs during intimal thickening and indicates that this is a result of Wnt4 upregulation.

The Wnt pathways in vascular disease: lessons from vascular development.

PURPOSE OF REVIEW: We aim to highlight the emerging evidence for the role of the Wnt signalling pathways in vascular disease and indicate how our current understanding is supported by observations of Wnt signalling in vascular development. RECENT FINDINGS: There is mounting direct and indirect evidence for an involvement of the Wnt pathways in multiple processes involved in atherogenesis. Although a systematic analysis of Wnt pathway in atherosclerosis has not been performed, it is apparent that altered expression of a handful of Wnt pathway proteins occurs in or regulates atherogenesis. Wnt pathways regulate endothelial dysfunction and vascular smooth muscle cell (VSMC) proliferation and migration and thereby intimal thickening. Furthermore, the Wnt pathways have the capacity to regulate inflammation and foam cell formation, pathological angiogenesis and calcification, which are crucial processes in plaque formation and stability. SUMMARY: A wealth of evidence has been presented for the involvement of the Wnt pathways in vascular development. Although less evidence exists for the regulation of vascular disease by the Wnt pathways, sufficient evidence exists to propose these pathways act as an important regulator of vascular disease. A greater understanding of Wnt pathways may reveal new therapeutic targets for vascular disease.

Haploinsufficiency of the germ cell-specific nuclear RNA binding protein hnRNP G-T prevents functional spermatogenesis in the mouse.

Human HNRNPGT, encoding the protein hnRNP G-T, is one of several autosomal retrogenes derived from RBMX. It has been suggested that HNRNPGT functionally replaces the sex-linked RBMX and RBMY genes during male meiosis. We show here that during normal mouse germ cell development, hnRNP G-T protein is strongly expressed during and after meiosis when proteins expressed from Rbmx or Rbmx-like genes are absent. Amongst these Rbmx-like genes, DNA sequence analyses indicate that two other mouse autosomal Rbmx-derived retrogenes have evolved recently in rodents and one already shows signs of degenerating into a non-expressed pseudogene. In contrast, orthologues of Hnrnpgt are present in all four major groups of placental mammals. The sequence of Hnrnpgt is under considerable positive selection suggesting it performs an important germ cell function in eutherians. To test this, we inactivated Hnrnpgt in ES cells and studied its function during spermatogenesis in chimaeric mice. Although germ cells heterozygous for this targeted allele could produce sperm, they did not contribute to the next generation. Chimaeric mice with a high level of mutant germ cells were infertile with low sperm counts and a high frequency of degenerate seminiferous tubules and abnormal sperm. Chimaeras made from a 1:1 mix of targeted and wild-type ES cell clones transmitted wild-type germ cells only. Our data show that haploinsufficiency of Hnrnpgt results in abnormal sperm production in the mouse. Genetic defects resulting in reduced levels of HNRNPGT could, therefore, be a cause of male infertility in humans.

The in vitro genotoxicity of orthopaedic ceramic (Al2O3) and metal (CoCr alloy) particles.

One of the biggest problems with orthopaedic joint replacements has been the tendency for metal-on-polyethylene implants to produce particulate wear debris. These particles stimulated adjacent macrophage infiltration, which caused destruction of bone and soft tissue, resulting in aseptic loosening of the implant. This problem led to the development of new implants with articulating surfaces that produce less volumetric wear (metal-on-metal, MOM, and ceramic-on-ceramic, COC). To determine whether there could be adverse biological effects from exposure to particulate wear debris after total hip replacement (THR), we investigated the in vitro genotoxic effects of alumina ceramic (Al(2)O(3)) particles in comparison with cobalt-chrome metal (CoCr alloy) particles. Primary human fibroblasts were exposed to Al(2)O(3) nanoparticles or CoCr alloy particles (0.1-10mg/T-75 flask) for 5 days. There were no significant differences in cell viability between control and ceramic-treated cells, at all doses and time-points studied. Cells exposed to CoCr alloy particles showed both dose- and time-dependent cytotoxicity. There was a small but significant increase in micronucleated binucleate cells after 24h of treatment with >1mg/T-75 flask of alumina particulates compared with controls, although no clear dose-response was observed. The induction of micronuclei was unaffected by the size or shape of the ceramic particles. The increase in micronucleated binucleate cells was much greater after exposure to CoCr particles for 24h, showing a clear dose-response curve. No increase in gamma-H2AX foci was noted in cells exposed to ceramic particles, in contrast with a significant increase of these foci in cells exposed to CoCr particles at comparable mass/surface doses. Cytogenetic analysis showed that both types of particle caused mainly numerical rather than structural chromosomal aberrations, with a greater number and variation of lesions induced by CoCr particles. In conclusion, our results show that alumina (Al(2)O(3)) ceramic particles are only weakly genotoxic to human cells in vitro when compared with metal (CoCr alloy) particles.

Zoledronate upregulates MMP-9 and -13 in rat vascular smooth muscle cells by inducing oxidative stress.

BACKGROUND: Bisphosphonates, including zoledronate, target osteoclasts and are widely used in the treatment of osteoporosis and other bone resorption diseases, despite side effects that include damaging the stomach epithelium. Beneficial and adverse effects on other organ systems, including the cardiovascular system, have also been described and could impact on the use of bisphosphonates as therapeutic agents. Vascular smooth muscle cells (VSMCs) are major constituents of the normal vascular wall and have a key role in intimal thickening and atherosclerosis, in part by secreting MMPs that remodel the extracellular matrix and cleave cell surface proteins or secreted mediators. In this study, we investigated the effects of zoledronate on MMP expression. METHODS: Rat VSMCs were stimulated by PDGF (50 ng/mL) plus TNF-α (10 ng/mL) or left unstimulated for a further 24 hours in serum-free medium. In other series of experiments, cells were pre-treated either with SC-514 (50 µM) or with apocynin (20 nM) for 2 hours, then zoledronate (100 µM) was added into 2% fetal calf serum containing medium for 24 hours. RESULTS AND DISCUSSION: Using isolated rat VSMCs in culture, zoledronate (100 µM) increased MMP-9 and -13 mRNA expressions but inhibited MMP-2 expression. MMP-9 and MMP-13 up-regulation was shown to depend on the NF-κB pathway; and this was activated by zoledronate. Furthermore, zoledronate elevated the levels of reactive oxygen species detected by either dichlorofluorescein in isolated VSMCs or lucigenin enhanced chemiluminescence in rat aortic rings in vitro. Apocynin, an inhibitor of NADPH oxidase, reversed NF-κB activation and MMP-9 and MMP-13 up-regulation by zoledronate. CONCLUSION: We conclude that zoledronate increases MMP-9 and MMP-13 expressions in rat VSMCs dependent upon stimulation of the NF-κB pathway by reactive oxygen species. Effects on MMP expression may contribute to the pharmacologic profile of bisphosphonates.

Plaque Size Is Decreased but M1 Macrophage Polarization and Rupture Related Metalloproteinase Expression Are Maintained after Deleting T-Bet in ApoE Null Mice.

BACKGROUND: Thelper1 (Th1) lymphocytes have been previously implicated in atherosclerotic plaque growth but their role in plaque vulnerability to rupture is less clear. We investigated whether T-bet knockout that prevents Th1 lymphocyte differentiation modulates classical (M1) macrophage activation or production of matrix degrading metalloproteinases (MMPs) and their tissue inhibitors, TIMPs. METHODS & RESULTS: We studied the effect of T-bet deletion in apolipoproteinE (ApoE) knockout mice fed a high fat diet (HFD) or normal chow diet (ND). Transcript levels of M1/M2 macrophage polarization markers, selected MMPs and TIMPs were measured by RT-qPCR in macrophages isolated from subcutaneous granulomas or in whole aortae. Immunohistochemistry of aortic sinus (AS) and brachiocephalic artery (BCA) plaques was conducted to quantify protein expression of the same factors. Deletion of T-bet decreased mRNA for the M1 marker NOS-2 in granuloma macrophages but levels of M2 markers (CD206, arginase-1 and Ym-1), MMPs-2, -9, -12, -13, -14 and -19 or TIMPs-1 to -3 were unchanged. No mRNA differences were observed in aortic extracts from mice fed a HFD for 12 weeks. Moreover, AS and BCA plaques were similarly sized between genotypes, and had similar areas stained for NOS-2, COX-2, MMP-12 and MMP-14 proteins. T-bet deletion increased MMP-13, MMP-14 and arginase-1 in AS plaques. After 35 weeks of ND, T-bet deletion reduced the size of AS and BCA plaques but there were no differences in the percentage areas stained for M1 or M2 markers, MMPs-12, -13, -14, or TIMP-3. CONCLUSIONS: Absence of Th1 lymphocytes is associated with reduced plaque size in ApoE knockout mice fed a normal but not high fat diet. In either case, M1 macrophage polarization and expression of several MMPs related to plaque instability are either maintained or increased.

Classical and Alternative Activation and Metalloproteinase Expression Occurs in Foam Cell Macrophages in Male and Female ApoE Null Mice in the Absence of T and B Lymphocytes.

BACKGROUND: Rupture of advanced atherosclerotic plaques accounts for most life-threatening myocardial infarctions. Classical (M1) and alternative (M2) macrophage activation could promote atherosclerotic plaque progression and rupture by increasing production of proteases, including matrix metalloproteinases (MMPs). Lymphocyte-derived cytokines may be essential for generating M1 and M2 phenotypes in plaques, although this has not been rigorously tested until now. METHODS AND RESULTS: We validated the expression of M1 markers (iNOS and COX-2) and M2 markers (arginase-1, Ym-1, and CD206) and then measured MMP mRNA levels in mouse macrophages during classical and alternative activation in vitro. We then compared mRNA expression of these genes ex vivo in foam cells from subcutaneous granulomas in fat-fed immune-competent ApoE knockout (KO) and immune-compromised ApoE/Rag-1 double-KO mice, which lack all T and B cells. Furthermore, we performed immunohistochemistry in subcutaneous granulomas and in aortic root and brachiocephalic artery atherosclerotic plaques to measure the extent of M1/M2 marker and MMP protein expression in vivo. Classical activation of mouse macrophages with bacterial lipopolysaccharide in vitro increased MMPs-13, -14, and -25 but decreased MMP-19 and TIMP-2 mRNA expressions. Alternative activation with IL-4 increased MMP-19 expression. Foam cells in subcutaneous granulomas expressed all M1/M2 markers and MMPs at ex vivo mRNA and in vivo protein levels, irrespective of Rag-1 genotype. There were also similar percentages of foam cell macrophages (FCMs) carrying M1/M2 markers and MMPs in atherosclerotic plaques from ApoE KO and ApoE/Rag-1 double-KO mice. CONCLUSION: Classical and alternative activation leads to distinct MMP expression patterns in mouse macrophages in vitro. M1 and M2 polarization in vivo occurs in the absence of T and B lymphocytes in either granuloma or plaque FCMs.

Regulation of VSMC behavior by the cadherin-catenin complex.

Vascular smooth muscle cells (VSMCs) are the predominant cell type within blood vessels. In normal vessels VSMC have low rates of proliferation, migration and apoptosis. However, increased VSMC proliferation, migration, and apoptosis rates radically alter the composition and structure of the blood vessel wall and contribute to vascular diseases such as atherosclerosis, in-stent restenosis and vein graft failure. Consequently, therapies that modulate VSMC proliferation, migration and apoptosis may be useful for treating vascular diseases. In this review article we discuss recently emerging research that has revealed that homophilic cell-cell contacts mediated by the cadherin:catenin complex and Wnt/beta-catenin signalling are important regulators of VSMC behaviour.