Deletion of bone marrow myeloperoxidase attenuates chronic kidney disease accelerated atherosclerosis.

Increased myeloperoxidase (MPO) expression and activity are associated with atherosclerotic disease in patients with chronic kidney disease (CKD). However, the causal relationship between MPO and the development and progression of atherosclerosis in patients with CKD is unknown. Eight-week-old male low-density-lipoprotein-receptor-deficient mice were subjected to 5/6 nephrectomy, irradiated, and transplanted with bone marrow from MPO-deficient mice to induce bone marrow MPO deletion (CKD-bMPOKO) or bone marrow from WT mice as a control to maintain preserved bone marrow MPO(CKD-bMPOWT). The mice were maintained on a high-fat/high-cholesterol diet for 16 weeks. As anticipated, both groups of mice exhibited all features of moderate CKD, including elevated plasma creatinine, lower hematocrit, and increased intact parathyroid hormone but did not demonstrate any differences between the groups. Irradiation and bone marrow transplantation did not further affect body weight, blood pressure, creatinine, or hematocrit in either group. The absence of MPO expression in the bone marrow and atherosclerotic lesions of the aorta in the CKD-bMPOKO mice was confirmed by immunoblot and immunohistochemistry, respectively. Decreased MPO activity was substantiated by the absence of 3-chlorotyrosine, a specific by-product of MPO, in aortic atherosclerotic lesions as determined by both immunohistochemistry and highly sensitive LC-MS. Quantification of the aortic lesional area stained with oil red O revealed that CKD-bMPOKO mice had significantly decreased aortic plaque area as compared with CKD-bMPOWT mice. This study demonstrates the reduction of atherosclerosis in CKD mice with the deletion of MPO in bone marrow cells, strongly implicating bone-marrow-derived MPO in the pathogenesis of CKD atherosclerosis.

Myeloperoxidase-derived oxidants damage artery wall proteins in an animal model of chronic kidney disease-accelerated atherosclerosis.

Increased myeloperoxidase (MPO) levels and activity are associated with increased cardiovascular risk among individuals with chronic kidney disease (CKD). However, a lack of good animal models for examining the presence and catalytic activity of MPO in vascular lesions has impeded mechanistic studies into CKD-associated cardiovascular diseases. Here, we show for the first time that exaggerated atherosclerosis in a pathophysiologically relevant CKD mouse model is associated with increased macrophage-derived MPO activity. Male 7-week-old LDL receptor-deficient mice underwent sham (control mice) or 5/6 nephrectomy and were fed either a low-fat or high-fat, high-cholesterol diet for 24 weeks, and the extents of atherosclerosis and vascular reactivity were assessed. MPO expression and oxidation products-protein-bound oxidized tyrosine moieties 3-chlorotyrosine, 3-nitrotyrosine, and o,o'-dityrosine-were examined with immunoassays and confirmed with mass spectrometry (MS). As anticipated, the CKD mice had significantly higher plasma creatinine, urea nitrogen, and intact parathyroid hormone along with lower hematocrit and body weight. On both the diet regimens, CKD mice did not have hypertension but had lower cholesterol and triglyceride levels than the control mice. Despite the lower cholesterol levels, CKD mice had increased aortic plaque areas, fibrosis, and luminal narrowing. They also exhibited increased MPO expression and activity (i.e. increased oxidized tyrosines) that co-localized with infiltrating lesional macrophages and diminished vascular reactivity. In summary, unlike non-CKD mouse models of atherosclerosis, CKD mice exhibit increased MPO expression and catalytic activity in atherosclerotic lesions, which co-localize with lesional macrophages. These results implicate macrophage-derived MPO in CKD-accelerated atherosclerosis.

Podocyte-specific JAK2 overexpression worsens diabetic kidney disease in mice.

Activation of JAK-STAT signaling has been implicated in the pathogenesis of diabetic kidney disease. An increased expression of JAK-STAT genes was found in kidney glomerular cells, including podocytes, in patients with early diabetic kidney disease. However, it is not known whether increased expression of JAK or STAT isoforms in glomerular cells can lead to worsening nephropathy in the setting of diabetes. Therefore, we overexpressed JAK2 mRNA specifically in glomerular podocytes of 129S6 mice to determine whether this change alone could worsen diabetic kidney disease. A 2-3 fold increase in glomerular JAK2 expression, an increase similar to that found in humans with early diabetic kidney disease, led to substantial and statistically significant increases in albuminuria, mesangial expansion, glomerulosclerosis, glomerular fibronectin accumulation, and glomerular basement membrane thickening, and a significant reduction in podocyte density in diabetic mice. Treatment with a specific JAK1/2 inhibitor for 2 weeks partly reversed the major phenotypic changes of diabetic kidney disease and specifically normalized expression of a number of downstream STAT3-dependent genes implicated in diabetic kidney disease progression. Thus, moderate increases in podocyte JAK2 expression at levels similar to those in patients with early diabetic kidney disease can lead directly to phenotypic and other alterations of progressive diabetic glomerulopathy. Hence, inhibition of these changes by treatment with a JAK1/2 inhibitor suggests that such treatment may help retard progression of early diabetic kidney disease in patients.

Digoxin-Mediated Upregulation of RGS2 Protein Protects against Cardiac Injury.

Regulator of G protein signaling (RGS) proteins have emerged as novel drug targets since their discovery almost two decades ago. RGS2 has received particular interest in cardiovascular research due to its role in regulating Gqsignaling in the heart and vascular smooth muscle. RGS2(-/-)mice are hypertensive, prone to heart failure, and display accelerated kidney fibrosis. RGS2 is rapidly degraded through the proteasome, and human mutations leading to accelerated RGS2 protein degradation correlate with hypertension. Hence, stabilizing RGS2 protein expression could be a novel route in treating cardiovascular disease. We previously identified cardiotonic steroids, including digoxin, as selective stabilizers of RGS2 protein in vitro. In the current study we investigated the functional effects of digoxin-mediated RGS2 protein stabilization in vivo. Using freshly isolated myocytes from wild-type and RGS2(-/-)mice treated with vehicle or low-dose digoxin (2µg/kg/day for 7 days) we demonstrated that agonist-induced cAMP levels and cardiomyocyte contractility was inhibited by digoxin in wild-type but not in RGS2(-/-)mice. This inhibition was accompanied by an increase in RGS2 protein levels in cardiomyocytes as well as in whole heart tissue. Furthermore, digoxin had protective effects in a model of cardiac injury in wild-type mice and this protection was lost in RGS2(-/-)mice. Digoxin is the oldest known therapy for heart failure; however, beyond its activity at the Na(+)/K(+)-ATPase, the exact mechanism of action is not known. The current study adds a novel mechanism, whereby through stabilizing RGS2 protein levels digoxin could exert its protective effects in the failing heart.

Divergent functions of the Rho GTPases Rac1 and Cdc42 in podocyte injury.

Podocytes are highly specialized epithelial cells with complex actin cytoskeletal architecture crucial for maintenance of the glomerular filtration barrier. The mammalian Rho GTPases Rac1 and Cdc42 are molecular switches that control many cellular processes, but are best known for their roles in the regulation of actin cytoskeleton dynamics. Here, we employed podocyte-specific Cre-lox technology and found that mice with deletion of Rac1 display normal podocyte morphology without glomerular dysfunction well into adulthood. Using the protamine sulfate model of acute podocyte injury, podocyte-specific deletion of Rac1 prevented foot process effacement. In a long-term model of chronic hypertensive glomerular damage, however, loss of Rac1 led to an exacerbation of albuminuria and glomerulosclerosis. In contrast, mice with podocyte-specific deletion of Cdc42 had severe proteinuria, podocyte foot process effacement, and glomerulosclerosis beginning as early as 10 days of age. In addition, slit diaphragm proteins nephrin and podocin were redistributed, and cofilin was dephosphorylated. Cdc42 is necessary for the maintenance of podocyte structure and function, but Rac1 is entirely dispensable in physiological steady state. However, Rac1 has either beneficial or deleterious effects depending on the context of podocyte impairment. Thus, our study highlights the divergent roles of Rac1 and Cdc42 function in podocyte maintenance and injury.

Inhibition of neutrophil extracellular trap formation alleviates vascular dysfunction in type 1 diabetic mice.

While neutrophil extracellular traps (NETs) have previously been linked to some diabetes-associated complications, such as dysfunctional wound healing, their potential role in diabetic vascular dysfunction has not been studied. Diabetic Akita mice were crossed with either Elane-/- or Pad4-/- mice to generate NET-deficient diabetic mice. By 24 weeks of age, Akita aortae showed markedly impaired relaxation in response to acetylcholine, indicative of vascular dysfunction. Both Akita-Elane-/- mice and Akita-Pad4-/- mice had reduced levels of circulating NETs and improved acetylcholine-mediated aortic relaxation. Compared with wild-type aortae, the thromboxane metabolite TXB2 was roughly 10-fold higher in both intact and endothelium-denuded aortae of Akita mice. In contrast, Akita-Elane-/- and Akita-Pad4-/- aortae had TXB2 levels similar to wild type. In summary, inhibition of NETosis by two independent strategies prevented the development of vascular dysfunction in diabetic Akita mice. Thromboxane was up-regulated in the vessel walls of NETosis-competent diabetic mice, suggesting a role for neutrophils in driving the production of this vasoconstrictive and atherogenic prostanoid.

Cardiotonic steroids stabilize regulator of G protein signaling 2 protein levels.

Regulator of G protein signaling 2 (RGS2), a G(q)-specific GTPase-activating protein, is strongly implicated in cardiovascular function. RGS2(-/-) mice are hypertensive and prone to heart failure, and several rare human mutations that accelerate RGS2 degradation have been identified among patients with hypertension. Therefore, pharmacological up-regulation of RGS2 protein levels might be beneficial. We used a ß-galactosidase complementation method to screen several thousand compounds with known pharmacological functions for those that increased RGS2 protein levels. Several cardiotonic steroids (CTSs), including ouabain and digoxin, increased RGS2 but not RGS4 protein levels. CTSs increased RGS2 protein levels through a post-transcriptional mechanism, by slowing protein degradation. RGS2 mRNA levels in primary vascular smooth muscle cells were unaffected by CTS treatment, whereas protein levels were increased 2- to 3-fold. Na(+)/K(+)-ATPase was required for the increase in RGS2 protein levels, because the effect was lost in Na(+)/K(+)-ATPase-knockdown cells. Furthermore, we demonstrated that CTS-induced increases in RGS2 levels were functional and reduced receptor-stimulated, G(q)-dependent, extracellular signal-regulated kinase phosphorylation. Finally, we showed that in vivo treatment with digoxin led to increased RGS2 protein levels in heart and kidney. CTS-induced increases in RGS2 protein levels and function might modify several deleterious mechanisms in hypertension and heart failure. This novel CTS mechanism might contribute to the beneficial actions of low-dose digoxin treatment in heart failure. Our results support the concept of small-molecule modulation of RGS2 protein levels as a new strategy for cardiovascular therapy.

Microarray analyses during adipogenesis: understanding the effects of Wnt signaling on adipogenesis and the roles of liver X receptor alpha in adipocyte metabolism.

Wnt signaling maintains preadipocytes in an undifferentiated state. When Wnt signaling is enforced, 3T3-L1 preadipocytes no longer undergo adipocyte conversion in response to adipogenic medium. Here we used microarray analyses to identify subsets of genes whose expression is aberrant when differentiation is blocked through enforced Wnt signaling. Furthermore, we used the microarray data to identify potentially important adipocyte genes and chose one of these, the liver X receptor alpha (LXR alpha), for further analyses. Our studies indicate that enforced Wnt signaling blunts the changes in gene expression that correspond to mitotic clonal expansion, suggesting that Wnt signaling inhibits adipogenesis in part through dysregulation of the cell cycle. Experiments designed to uncover the potential role of LXR alpha in adipogenesis revealed that this transcription factor, unlike CCAAT/enhancer binding protein alpha and peroxisome proliferator-activated receptor gamma, is not adipogenic but rather inhibits adipogenesis if inappropriately expressed and activated. However, LXR alpha has several important roles in adipocyte function. Our studies show that this nuclear receptor increases basal glucose uptake and glycogen synthesis in 3T3-L1 adipocytes. In addition, LXR alpha increases cholesterol synthesis and release of nonesterified fatty acids. Finally, treatment of mice with an LXR alpha agonist results in increased serum levels of glycerol and nonesterified fatty acids, consistent with increased lipolysis within adipose tissue. These findings demonstrate new metabolic roles for LXR alpha and increase our understanding of adipogenesis.

GLUT4 facilitative glucose transporter specifically and differentially contributes to agonist-induced vascular reactivity in mouse aorta.

OBJECTIVE: We hypothesized that GLUT4 is a predominant facilitative glucose transporter in vascular smooth muscle cells (VSMCs), and GLUT4 is necessary for agonist-induced VSMC contraction. METHODS AND RESULTS: Glucose deprivation and indinavir, a GLUT4 antagonist, were used to assess the role of GLUT4 and non-GLUT4 transporters in vascular reactivity. In isolated endothelium-denuded mouse aorta, approximately 50% of basal glucose uptake was GLUT4-dependent. Norepinephrine-mediated contractions were dependent on both GLUT4 and non-GLUT4 transporters, serotonin (5-HT)-mediated contractions were mainly GLUT4-dependent, and prostaglandin (PG) F(2alpha)-mediated contractions were dependent on non-GLUT4 transporters, whereas indinavir had no effect in GLUT4 knockout vessels. We also observed a 46% decrease in GLUT4 expression in aortas from angiotensin II hypertensive mice. Indinavir caused a less profound attenuation of maximal 5-HT-mediated contraction in these vessels, corresponding to the lower GLUT4 levels in the hypertensive aortas. Finally, and somewhat surprisingly, chronic GLUT4 knockout was associated with increased vascular reactivity compared with that in wild-type animals, suggesting that chronic absence or reduction of GLUT4 expression in VSMCs leads to opposite effects observed with acute inhibition of GLUT4. CONCLUSIONS: Thus, we conclude that GLUT4 is constitutively expressed in large arteries and likely participates in basal glucose uptake. In addition, GLUT4, as well as other non-GLUT4 facilitative glucose transporters, are necessary for agonist-induced contraction, but each transporter participates in VSMC contraction selectively, depending on the agonist, and changes in GLUT4 expression may account for some of the functional changes associated with vascular diseases like hypertension.

Maintenance of GLUT4 expression in smooth muscle prevents hypertension-induced changes in vascular reactivity.

Previous studies have shown that expression of GLUT4 is decreased in arterial smooth muscle of hypertensive rats and mice and that total body overexpression of GLUT4 in mice prevents enhanced arterial reactivity in hypertension. To demonstrate that the effect of GLUT4 overexpression on vascular responses is dependent on vascular smooth muscle GLUT4 rather than on some systemic effect we developed and tested smooth-muscle-specific GLUT4 transgenic mice (SMG4). When made hypertensive with angiotensin II, both wild-type and SMG4 mice exhibited similarly increased systolic blood pressure. Responsiveness to phenylephrine, serotonin, and prostaglandin F2α was significantly increased in endothelium-intact aortic rings from hypertensive wild-type mice but not in aortae of SMG4 mice. Inhibition of Rho-kinase equally reduced serotonin-stimulated contractility in aortae of hypertensive wild-type and SMG4-mice. In addition, acetylcholine-stimulated relaxation was significantly decreased in aortic rings of hypertensive wild-type mice, but not in rings of SMG4 mice. Inhibition of either prostacylin receptors or cyclooxygenase-2 reduced relaxation in rings of hypertensive SMG4 mice. Inhibition of cyclooxygenase-2 had no effect on relaxation in rings of hypertensive wild-type mice. Cyclooxygenase-2 protein expression was decreased in hypertensive wild-type aortae but not in hypertensive SMG4 aortae compared to nonhypertensive controls. Our results demonstrate that smooth muscle expression of GLUT4 exerts a major effect on smooth muscle contractile responses and endothelium-dependent vasorelaxation and that normal expression of GLUT4 in vascular smooth muscle is required for appropriate smooth muscle and endothelial responses.

A rapid, PPAR-gamma-dependent effect of pioglitazone on the phosphorylation of MYPT.

Peroxisome proliferator-activated receptor (PPAR)-gamma ligands, thiazolidinediones, have been demonstrated to regulate vascular reactivity. We examined the effect of pioglitazone (PIO; 20 muM) in rat primary cultured aortic smooth muscle cells on constitutive phosphorylation of the regulatory subunit of myosin phosphatase (MYPT). PIO decreased the phosphorylation of Thr(697) on MYPT within 15 min, and the inhibition was maintained up to 6 h. The PPAR-gamma antagonist GW-9662 (5 microM) abrogated the inhibition of Thr(697) phosphorylation mediated by PIO. Because longer-term PIO treatment inhibits RhoA/Rho kinase (ROCK) signaling and Thr(697) phosphorylation, we tested the effect of the ROCK inhibitor Y-27632 (10 muM) on the inhibition of Thr(697) phosphorylation by PIO. Y-27632 alone inhibited Thr(697) phosphorylation, and there was an additive effect with PIO. In addition, up to 1 h of PIO treatment did not affect RhoA localization or decrease ROCK-dependent phosphorylation of Thr(855). These results suggest that the effect of PIO is independent of inhibition of RhoA/ROCK. PIO increased the phosphorylation of Ser(696) in the same time course as its effect on Thr(697). Ser(696) has been shown to be phosphorylated by PKA and PKG. PKA inhibitor H-89 (10 microM) and PKG inhibitor KT-5823 (0.5 microM) abrogated the effect of PIO on both Thr(697) and Ser(696) phosphorylation. The constitutive turnover of phosphorylation of Thr(697) is rapid, suggesting that the decreased phosphorylation of Thr(697) by PIO is due to enhanced phosphorylation of Ser(696). This is supported by the finding that PIO blocks ANG II-stimulated phosphorylation of Thr(697) but not ANG II-stimulated RhoA translocation. Therefore, the effect of shorter-term PIO apparently is to increase myosin light chain phosphatase activity, thereby desensitizing the vascular smooth muscle to agonist signaling.

Preserved expression of GLUT4 prevents enhanced agonist-induced vascular reactivity and MYPT1 phosphorylation in hypertensive mouse aorta.

We previously showed that GLUT4 expression is decreased in arterial smooth muscle of deoxycorticosterone acetate (DOCA)-salt hypertensive rats and that GLUT4-knockout mice have enhanced arterial reactivity. Therefore, we hypothesized that increased GLUT4 expression in vascular smooth muscle in vivo would prevent enhanced arterial reactivity and possibly reduce blood pressure in DOCA-salt hypertensive mice. Adult wild-type (WT) and GLUT4 transgenic (TG) mice were subjected to DOCA-salt hypertension with uninephrectomy or underwent uninephrectomy and remained normotensive. GLUT4 expression was increased more than twofold in the aortas of GLUT4 TG mice compared with WT aortas. Eight weeks after implantation of the DOCA pellets, GLUT4 expression decreased by 75% in aortas of WT hypertensive mice, but not in GLUT4 TG hypertensive aortas. Systolic blood pressure was significantly and similarly increased in WT and GLUT4 TG DOCA-salt mice compared with their respective sham-treated controls (159 vs. 111 mmHg). Responsiveness to the contractile agonist 5-HT was significantly increased in aortic rings from WT DOCA-salt mice but remained normal in GLUT4 TG DOCA mice. Phosphorylation of the myosin phosphatase targeting subunit MYPT1 was significantly enhanced in aortas of WT DOCA-salt mice, and this increase was prevented in GLUT4 TG mice. MYPT1 phosphorylation was also increased in nonhypertensive GLUT4-knockout mice. Myosin phosphatase, a major negative regulator of calcium sensitivity, is itself negatively regulated by phosphorylation of MYPT1. Therefore, our results show that preservation of GLUT4 expression prevents enhanced arterial reactivity in hypertension, possibly via effects on myosin phosphatase activity.

Effects of PPAR-gamma ligands on vascular smooth muscle marker expression in hypertensive and normal arteries.

Having previously demonstrated that glucose transporter-4 (GLUT4) expression was reduced in aortas and carotid arteries of deoxycorticosterone acetate (DOCA) salt-hypertensive rats, we hypothesized that troglitazone (TG), through activation of peroxisome proliferator-activated receptor-gamma (PPAR-gamma), would stabilize GLUT4 expression and possibly preserve the differentiated phenotype in vascular smooth muscle cells. In DOCA salt-hypertensive rats treated with TG (100 mg/day), there was a significant (P < 0.001) decrease in systolic blood pressure (BP; 149.9 +/- 4.4 mmHg) compared with the untreated DOCA salt-hypertensive rats (202.2 +/- 10.34 mmHg). Separate trials with rosiglitazone (RS; 3 mg/day) demonstrated a significant (P < 0.001) decrease in BP (DOCA salt, 164.2 +/- 9.8 vs. DOCA-RS, 124.9 +/- 3.7 mmHg) comparable to that with TG. Expression of GLUT4, h-caldesmon, and smooth muscle myosin heavy chain SM2 was significantly decreased in aortas of DOCA salt-hypertensive rats and was reversed by TG to levels similar to those in aortas of sham-treated rats. TG (50 microM) induced GLUT4 and h-caldesmon expression in 24-h culture of explanted carotid arteries of DOCA salt-hypertensive rats, and the endogenous PPAR-gamma ligand 15-deoxy-Delta(12-14)-prostaglandin J(2) (PGJ(2); 20 microM) and TG (50 microM) similarly increased GLUT4, h-caldesmon, and SM2 protein expression in explanted aortas. The expression of activated, phosphorylated Akt was increased by PGJ(2) and TG with no significant effect on total Akt levels. Inhibition of phosphorylated Akt expression using the phosphatidylinositol 3-kinase inhibitor LY-294002 (16 microM) abrogated the increased expression of h-caldesmon and SM2. These data demonstrate that PPAR-gamma agonists maintain or induce expression of markers of the contractile phenotype independently of their effects on hypertension, and that this effect may be mediated through activation of phosphatidylinositol 3-kinase/Akt.