Lead Decreases Bone Morphogenetic Protein-7 (BMP-7) Expression and Increases Renal Cell Carcinoma Growth in a Sex-Divergent Manner.

Both tissue and blood lead levels are elevated in renal cell carcinoma (RCC) patients. These studies assessed the impact of the subchronic lead challenge on the progression of RCC in vitro and in vivo. Lead challenge of Renca cells with 0.5 µM lead acetate for 10 consecutive passages decreased E-cadherin expression and cell aggregation. Proliferation, colony formation, and wound healing were increased. When lead-challenged cells were injected into mice, tumor size at day 21 was increased; interestingly, this increase was seen in male but not female mice. When mice were challenged with 32 ppm lead in drinking water for 20 weeks prior to tumor cell injection, there was an increase in tumor size in male, but not female, mice at day 21. To investigate the mechanism underlying the sex differences, the expression of sex hormone receptors in Renca cells was examined. Control Renca cells expressed estrogen receptor (ER) alpha but not ER beta or androgen receptor (AR), as assessed by qPCR, and the expression of ERα was increased in tumors in both sexes. In tumor samples harvested from lead-challenged cells, both ERα and AR were detected by qPCR, yet there was a significant decrease in AR seen in lead-challenged tumor cells from male mice only. This was paralleled by a plate-based array demonstrating the same sex difference in BMP-7 gene expression, which was also significantly decreased in tumors harvested from male but not female mice; this finding was validated by immunohistochemistry. A similar expression pattern was seen in tumors harvested from the mice challenged with lead in the drinking water. These data suggest that lead promotes RCC progression in a sex-dependent via a mechanism that may involve sex-divergent changes in BMP-7 expression.

Adventitial macrophage accumulation impairs perivascular nerve function in mesenteric arteries with inflammatory bowel disease.

Introduction: Inflammatory bowel disease involves aberrant immune responses and is associated with both cardiovascular disease risk and altered intestinal blood flow. However, little is known about how inflammatory bowel disease affects regulation of perivascular nerves that mediate blood flow. Previous work found perivascular nerve function is impaired in mesenteric arteries with Inflammatory bowel disease. The purpose of this study was to determine the mechanism of impaired perivascular nerve function. Methods: RNA sequencing was performed on mesenteric arteries from IL10-/- mice treated with H. hepaticus to induce disease (inflammatory bowel disease) or left non-gavaged (Control). For all other studies, Control and Inflammatory bowel disease mice received either saline or clodronate liposome injections to study the effect of macrophage depletion. Perivascular nerve function was assessed using pressure myography and electrical field stimulation. Leukocyte populations, and perivascular nerves, and adventitial neurotransmitter receptors were labeled using fluorescent immunolabeling. Results: Inflammatory bowel disease was associated with increases in macrophage-associated gene expression, and immunolabeling showed accumulation of adventitial macrophages. Clodronate liposome injection eliminated adventitial macrophages, which reversed significant attenuation of sensory vasodilation, sympathetic vasoconstriction and sensory inhibition of sympathetic constriction in inflammatory bowel disease. Acetylcholine-mediated dilation was impaired in inflammatory bowel disease and restored after macrophage depletion, but sensory dilation remained nitric oxide independent regardless of disease and/or macrophage presence. Conclusion: Altered neuro-immune signaling between macrophages and perivascular nerves in the arterial adventitia contributes to impaired vasodilation, particularly via dilatory sensory nerves. Targeting the adventitial macrophage population may help preserve intestinal blood flow in Inflammatory bowel disease patients.

Multifaceted role of plasminogen activator inhibitor-1 in regulating early remodeling of vein bypass grafts.

OBJECTIVE: The role of plasminogen activator inhibitor-1 (PAI-1) in vein graft (VG) remodeling is undefined. We examined the effect of PAI-1 on VG intimal hyperplasia and tested the hypothesis that PAI-1 regulates VG thrombin activity. METHODS AND RESULTS: VGs from wild-type (WT), Pai1(-/-), and PAI-1-transgenic mice were implanted into WT, Pai1(-/-), or PAI-1-transgenic arteries. VG remodeling was assessed 4 weeks later. Intimal hyperplasia was significantly greater in PAI-1-deficient mice than in WT mice. The proliferative effect of PAI-1 deficiency was retained in vitronectin-deficient mice, suggesting that PAI-1's antiproteolytic function plays a key role in regulating intimal hyperplasia. Thrombin-induced proliferation of PAI-1-deficient venous smooth muscle cells (SMC) was significantly greater than that of WT SMC, and thrombin activity was significantly higher in PAI-1-deficient VGs than in WT VGs. Increased PAI-1 expression, which has been associated with obstructive VG disease, did not increase intimal hyperplasia. CONCLUSIONS: Decreased PAI-1 expression (1) promotes intimal hyperplasia by pathways that do not require vitronectin and (2) increases thrombin activity in VG. PAI-1 overexpression, although it promotes SMC migration in vitro, did not increase intimal hyperplasia. These results challenge the concept that PAI-1 drives nonthrombotic obstructive disease in VG and suggest that PAI-1's antiproteolytic function, including its antithrombin activity, inhibits intimal hyperplasia.

C-reactive protein enhances tissue factor expression by vascular smooth muscle cells: mechanisms and in vivo significance.

OBJECTIVE: We examined the impact of C-reactive protein (CRP) on vascular smooth muscle cell (VSMC) expression of tissue factor (TF) and TF pathway inhibitor (TFPI). METHODS AND RESULTS: TF mRNA, protein, and activity levels were significantly higher in VSMCs isolated from CRP-transgenic (Tg) mice than from wild-type (WT) mice. TFPI expression was significantly downregulated in CRP-Tg versus WT VSMCs. Transfection of human VSMCs with CRP expression plasmid significantly increased TF expression and decreased TFPI expression. Gene silencing of Fc gamma receptor IIIa (Fc gammaRIIIa) blocked the effect of CRP on VSMC TF expression. CRP activated p44/42, but not p38 or JNK MAP kinase (MAPK), and the effect of CRP on TF expression was blocked by pharmacological inhibitor of p44/42, but not p38 or JNK MAPK. Reactive oxygen species (ROS) scavengers blocked CRP-induced upregulation of VSMC TF expression. In vivo analyses revealed significant increases in TF expression and decreases in TFPI expression in carotid arteries of CRP-Tg mice versus WT mice. CONCLUSIONS: CRP increases TF and decreases TFPI expression by VSMCs in vitro and in vivo. Induction of TF expression by CRP is mediated by Fc gammaRIIIa, p44/42 MAPK, and ROS generation. These data offer important insights into the role of CRP in the pathogenesis of arterial thrombosis.

Double knockouts reveal that protein tyrosine phosphatase 1B is a physiological target of calpain-1 in platelets.

Calpains are ubiquitous calcium-regulated cysteine proteases that have been implicated in cytoskeletal organization, cell proliferation, apoptosis, cell motility, and hemostasis. Gene targeting was used to evaluate the physiological function of mouse calpain-1 and establish that its inactivation results in reduced platelet aggregation and clot retraction potentially by causing dephosphorylation of platelet proteins. Here, we report that calpain-1 null (Capn1-/-) platelets accumulate protein tyrosine phosphatase 1B (PTP1B), which correlates with enhanced tyrosine phosphatase activity and dephosphorylation of multiple substrates. Treatment of Capn1-/- platelets with bis(N,N-dimethylhydroxamido)hydroxooxovanadate, an inhibitor of tyrosine phosphatases, corrected the aggregation defect and recovered impaired clot retraction. More importantly, platelet aggregation, clot retraction, and tyrosine dephosphorylation defects were rescued in the double knockout mice lacking both calpain-1 and PTP1B. Further evaluation of mutant mice by the ferric chloride-induced arterial injury model suggests that the Capn1-/- mice are relatively resistant to thrombosis in vivo. Together, our results demonstrate that PTP1B is a physiological target of calpain-1 and suggest that a similar mechanism may regulate calpain-1-mediated tyrosine dephosphorylation in other cells.