Peroxisome proliferator‐activated receptor‐γ (PPARγ) is a ligand‐activated transcriptional regulator of genes encoding proteins involved in adipogenesis, lipid metabolism and glucose homeostasis. ...Synthetic ligands of PPARγ (thiazolidinediones such as pioglitazone) are utilized for improving insulin resistance. PPARγ ligands have been shown to inhibit the development of atherosclerosis in murine models of this disease, but their effect on preexisting atherosclerosis is unknown. We tested the hypothesis that pioglitazone could reverse atherosclerosis in LDL‐receptor null (LDLR−/−) mice.
Methods
LDLR−/− mice (5 week old males) were divided into 3 groups (20 mice/group) and fed a high fat diet for 8 weeks. At this time 1/3 of the mice were sacrificed to establish a baseline of atherosclerotic lesion development. The remaining mice were continued on a high fat diet and half were given pioglitazone (15mg/kg/day) in the diet for 6 more weeks prior to sacrifice. Aortas were dissected and neutral lipid was stained with oil‐red‐o. Lipid stained lesion areas were quantified. rtPCR was performed using peritoneal macrophage RNA to quantify mRNA of efflux proteins, ABCA1/G1.
Results
Lesion area in pioglitazone‐treated mice was 36% less (p<0.05) than in mice fed with high‐fat diet alone for 14 weeks, but not different from mice fed with diet alone for 8 weeks. Blood glucose and lipid profiles did not differ between the three groups. Results of gene expression studies are pending.
Conclusion
Pioglitazone inhibited progression of atherosclerosis but did not decrease the size of pre‐existing lesions. Studies to evaluate the effect of longer‐term treatment are in progress.
Supported by NHLBI PO1‐HL072942/The Silbermann and Abercrombie Foundations
Glucocorticoid regulation of proteoglycan synthesis in mesangial cells.
Proteoglycans are integral components of the mesangial matrix and glomerular permeability barrier. Recent studies have shown ...that changes in glomerular proteoglycan expression may play a major role in the pathogenesis of renal disease. Steroid hormones are used as first-choice therapy for the treatment of glomerular diseases, however, the effects of glucocorticoids on expression of glomerular proteoglycans are unknown.
This study examined the effects of in vitro and in vivo administration of dexamethasone on proteoglycan synthesis and gene expression of proteoglycan core proteins using rat (RMC) and human (HMC) mesangial cells.
Treatment of cultured RMC with dexamethasone resulted in a dose- and time-dependent decrease (P < 0.05) in both cell-associated and secreted proteoglycan synthesis to approximately 50% of control levels. This effect was inhibited by the glucocorticoid antagonist mifepristone, and mimicked by prednisolone or corticosterone treatment. Separation of proteoglycans by ion-exchange and gel permeation chromatography suggested that chondrotin sulfate/dermatan sulfate proteoglycans were down-regulated after steroid treatment. Northern blot analysis, RT-PCR, Western blot, and promoter activity assays revealed that dexamethasone caused a significant decrease in decorin mRNA (to 61 ± 8% of controls), whereas biglycan expression and promoter activity were increased after steroid treatment. A similar trend was found in glomeruli isolated from rats treated in vivo with dexamethasone.
These results demonstrate that treatment of mesangial cells with steroids results in a decrease in total proteoglycan synthesis, as well as subtype-specific changes in proteoglycan core protein gene expression by transcriptional control, furthering our understanding of the effects of steroid treatment on the renal glomeruli.
Recently, we have shown that treatment of stroke-prone spontaneously hypertensive rats with angiotensin inhibitors for a limited time-window before puberty results in an attenuation of hypertensive ...nephrosclerosis in later life. The aim of this study was to examine the applicability of this therapeutic paradigm to a low-renin model. Dahl salt-sensitive (Dahl-S) rats were fed a high-salt diet from age 6 weeks. Some rats were treated with the angiotensin receptor blocker (ARB) candesartan cilexetil (2 mg/kg/d) from weaning to puberty (age 3-10 weeks), whereas other rats were treated continuously until overt renal damage was seen (age 3-16 weeks). Dahl-S rats on a high salt diet had increased blood pressure (207 +/- 3 vs. 125 +/- 2 mm Hg), proteinuria, and glomerular/vascular histological changes. The prepubertal treatment with ARB resulted in a continued suppression of blood pressure (153 +/- 2 mm Hg) at 16 weeks. Both proteinuria and renal histological changes were significantly (p < 0.05) attenuated, but not completely prevented by the treatment. No significant differences in plasma renin activity, renin mRNA, or AT1/AT2 mRNA were seen between groups. These results suggest that prepubertal treatment affords sustained renoprotection, even in an animal model with a suppressed renin-angiotensin system, and support the notion that appropriate prepubertal intervention may lead to a partial attenuation in the susceptibility to inherited renal diseases.
Receptors with a heptahelical structure initiate signal transduction by interacting with specific Gα proteins. The aim of this study was to analyze the ability of type 1 (AT1) and type 2 (AT2) ...angiotensin receptors to recognize the receptor coupling regions of Gα proteins using our previously described technique (Ikezu, T., Okamoto, T., Komatsuzaki, K., Matsui, T., Martyn, J.A.J., Nishimoto, I., 1996. Negative transactivation of cAMP response element by familial Alzheimer's mutants of APP. EMBO J. 15, 2468–2475; Komatsuzaki, K., Murayama, Y., Giambarella, U., Ogata, E., Seino, S., Nishimoto, I., 1996. A novel system that reports the G-proteins linked to a given receptor: a study of the type 3 somatostatin receptor. FEBS Lett. 406, 165–170). Chimeric Gαs protein constructs, whose receptor binding regions contained sequences from the four major families of Gα proteins (Gαq, Gαi, Gα12, Gαs), were cotransfected with AT1 or AT2 receptors in COS cells, then stimulated with angiotensin II (Ang II). Changes in cellular cAMP were assayed on cell lysates by enzyme immunoassay. In the case of the Gαq family, cotransfection of AT1 with Gα11/Gαs, Gα14/Gαs, Gα16/Gαs, elicited significant increases in cAMP after agonist stimulation. Confirmatory results were found using an independent
35SGTPγS binding assay. Further examination using chimeric G proteins for Gα12 proteins and Gαi family proteins provided evidence that the AT1 receptor can recognize sequences from Gα12, Gαi1/i2, Gαz, Gαo, while both receptors interacted with Gαi3. These results provide a Gα protein recognition database for both AT1 and AT2 receptors, which may be important for understanding the full spectrum of cellular responses mediated by the hormone Ang II.
Thiazolidinediones, a class of drugs for the treatment of type-2 diabetes, are synthetic ligands for peroxisome proliferator-activated receptor-gamma. They have been demonstrated to possess ...cardioprotective effects in humans and anti-atherogenic properties in animal models. However, the question remains whether a peroxisome proliferator-activated receptor-gamma ligand can reverse the development of atherosclerosis. In this study, we tested the effects of pioglitazone on the development of established atherosclerosis in low-density lipoprotein receptor-null mice. We observed that atherosclerosis in low-density lipoprotein receptor-null mice progressed when mice were fed a high-fat diet. Pioglitazone treatment of atherogenic mice prevented this progression of atherosclerosis from its middle stages of disease, but was not able to reverse it. Withdrawal of the high-fat diet from mice with advanced atherosclerosis did not result in a reduction in lesion sizes. Pioglitazone treatment also had no effect on advanced atherosclerosis. Levels of high density lipoprotein cholesterol correlated inversely with lesion development when pioglitazone was given during lesion progression. However, pioglitazone had no effect on circulating high density lipoprotein levels in mice in which treatment was initiated following 14 weeks on the high-fat diet. These findings have implications for the analysis of therapeutic agents in murine models of atherosclerosis and the use of pioglitazone in patients with established atherosclerosis.
Abstract only P84 Previously, we and others have shown that angiotensin II enhances vascular smooth muscle cell extracellular matrix synthesis via stimulation of the type 1 angiotensin (AT1) ...receptor. Recently, expression of the type 2 (AT2) receptor has been confirmed in the adult vasculature, but its role in vascular remodeling has not yet been fully defined. In particular, conflicting data from in vivo studies have reported that AT2 receptor inhibition may either attenuate or enhance vascular hypertrophy and fibrosis. The aim of this study was to clarify the effects of direct stimulation of AT2 receptors on collagen synthesis in vascular smooth muscle cells in vitro. Firstly, retroviral gene transfer was used to supplement adult vascular smooth muscle cells with AT2 receptors to mimic the vasculature in vivo. Treatment of these cells with the AT2 receptor agonist CGP42212A (10-7 mol/L) alone did not cause a significant change in p42/p44 MAP kinase activity, but caused a modest (33%) decrease in protein tyrosine phosphatase activity. Treatment with CGP42112A also caused a dose- and time-dependent increase in both cell-associated and secretory collagen synthesis (148+17% of control at 48 h, p<0.05) which was completely inhibited by the AT2 receptor antagonist PD123319, but unaffected by the AT1 receptor antagonist losartan. The AT2 receptor-mediated stimulation of collagen synthesis was unaffected by tyrosine phosphatase inhibitors sodium orthovanadate and okadaic acid, but attenuated by pretreatment with pertussis toxin or Galphai antisense oligonuclotides. These results suggest that direct AT2 receptor stimulation can increase rather than decrease collagen synthesis in vascular smooth muscle cells, and suggest a role for Galphai in the AT2 receptor-mediated effects.
Control of adrenal aldosterone secretion is an important endocrine mechanism mediated by angiotensin II (Ang. II). Recently three subtypes of angiotensin receptors have been demonstrated in the rat ...adrenal. Our aim was to examine if these receptors are affected by hormone treatment in vivo. Treatment of rats with ACTH resulted in a decrease in AT2 receptor binding from 766±95 to 310±51 fmol/mg protein (
P<0.05), without significant changes in AT1 receptors. AT2 receptor mRNA was also decreased (18±2% of control,
P<0.05; 25±7% of control,
P<0.05) after both 2 and 7 day treatment with ACTH. Changes in ATla receptor mRNA or total AT1 mRNA did not reach statistical significance. Moreover, treatment with dexamethasone or aldosterone did not affect AT1a, AT1b, or AT2 mRNA. These results demonstrate that ACTH treatment results in subtype-specific changes in adrenal AT receptor gene expression in vivo.
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