Ryoko Shimizu-Hirota

Angiotensin II Type 2 Receptors Stimulate Collagen Synthesis in Cultured Vascular Smooth Muscle Cells

Previously, we and others have shown that angiotensin II enhances vascular smooth muscle cell extracellular matrix synthesis via stimulation of the angiotensin II type 1 (AT1) receptor. Recently, expression of the type 2 (AT2) receptor has been confirmed in the adult vasculature, but its role has not yet been fully defined. The aim of the present study was to examine the effects of stimulation of AT2 receptors on collagen synthesis in vascular smooth muscle cells. Retroviral gene transfer was used to supplement adult vascular smooth muscle cells with AT2 receptors to mimic the vasculature in vivo. The 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 (30% to 50%) 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 hours, P <0.05), which was completely inhibited by the AT2 receptor antagonist PD123319, unaffected by the AT1 receptor antagonist losartan, and attenuated by treatment with pertussis toxin or G&agr;i antisense oligonucleotides. Interestingly, studies in other cell lines demonstrated that CGP42112A caused similar results in transfected mesangial cells but had essentially opposite effects in fibroblasts (NIH-3T3-AT2). These results suggest that AT2 receptor stimulation can increase collagen synthesis in vascular smooth muscle cells via a G&agr;i-mediated mechanism and provide evidence for heterogeneity in the effects of AT2 receptor stimulation in different tissues.

Neutrophil-Derived Matrix Metalloproteinase 9 Triggers Acute Aortic Dissection

Background— Acute aortic dissection (AAD) is a life-threatening vascular disease without effective pharmaceutical therapy. Matrix metalloproteinases (MMPs) are implicated in the development of chronic vascular diseases including aneurysm, but the key effectors and mechanism of action remain unknown. To define further the role of MMPs in AAD, we screened circulating MMPs in AAD patients, and then generated a novel mouse model for AAD to characterize the mechanism of action. Methods and Results— MMP9 and angiotensin II were elevated significantly in blood samples from AAD patients than in those from the patients with nonruptured chronic aortic aneurysm or healthy volunteers. Based on the findings, we established a novel AAD model by infusing angiotensin II to immature mice that had been received a lysyl oxidase inhibitor, &bgr;-aminopropionitrile monofumarate. AAD was developed successfully in the thoracic aorta by angiotensin II administration to &bgr;-aminopropionitrile monofumarate-treated wild-type mice, with an incidence of 20%, 80%, and 100% after 6, 12, and 24 hours, respectively. Neutrophil infiltrations were observed in the intima of the thoracic aorta, and the overexpression of MMP9 in the aorta was demonstrated by reverse transcription polymerase chain reaction, gelatin zymography, and immunohistochemistry. The incidence of AAD was reduced significantly by 40% following the administration of an MMP inhibitor and was almost blocked completely in MMP−/− mice without any influence on neutrophil infiltration. Neutrophil depletion by injection of anti-granulocyte-differentiation antigen-1 (anti-Gr-1) antibody also significantly decreased the incidence of AAD. Conclusions— These data suggest that AAD is initiated by neutrophils that have infiltrated the aortic intima and released MMP9 in response to angiotensin II.

Extracellular Matrix Glycoprotein Biglycan Enhances Vascular Smooth Muscle Cell Proliferation and Migration

Abstract— Proteoglycans are produced and secreted by vascular smooth muscle cells, but the pathophysiological role of these glycoproteins in the vasculature is an enigma. Because the small leucine-rich proteoglycan (SLRP) biglycan is overexpressed in arteriosclerotic lesions, we produced mice constitutively overexpressing biglycan in the vascular smooth muscle, in order to examine the effects on vascular pathology. In the aorta and renal vasculature, increased vascular proliferation was seen both in the basal state and after infusion of angiotensin II (Ang II) in the transgenic mice compared with wild-type controls. In addition, the combination of biglycan overexpression and Ang II infusion resulted in marked increases in vascular smooth muscle cell proliferation and migration in the coronary arteries, as well as increases in fibrosis surrounding the vessels. In vitro, biglycan caused an increase in thymidine incorporation and migration of vascular smooth muscle cells, whereas these parameters were unchanged or reduced in endothelial cells. Moreover, addition of biglycan resulted in an increase in cdk2 expression and decrease in p27 levels in the vascular smooth muscle cells. These results suggest that this extracellular matrix SLRP may be involved in the regulation of vascular smooth muscle growth and migration through cdk2- and p27-dependent pathways. Furthermore, changes in biglycan expression could be a factor influencing the susceptibility of arteries to vascular injury, and may play a direct role in the pathogenesis of vascular lesions.

Adventitial CXCL1/G-CSF expression in response to acute aortic dissection triggers local neutrophil recruitment and activation leading to aortic rupture

Rationale: In-hospital outcomes are generally acceptable in patients with type B dissection; however, some patients present with undesirable complications, such as aortic expansion and rupture. Excessive inflammation is an independent predictor of adverse clinical outcomes. Objective: We have investigated the underlying mechanisms of catastrophic complications after acute aortic dissection (AAD) in mice. Methods and Results: When angiotensin II was administered in lysyl oxidase inhibitor–preconditioned mice, AAD emerged within 24 hours. The dissection was initiated at the proximal site of the descending thoracic aorta and propagated distally into an abdominal site. Dissection of the aorta caused dilatation, and ≈70% of the mice died of aortic rupture. AAD triggered CXCL1 and granulocyte-colony stimulating factor expression in the tunica adventitia of the dissected aorta, leading to elevation of circulating CXCL1/granulocyte-colony stimulating factor levels. Bone marrow CXCL12 was reduced. These chemokine changes facilitated neutrophil egress from bone marrow and infiltration into the aortic adventitia. Interference of CXCL1 function using an anti-CXCR2 antibody reduced neutrophil accumulation and limited aortic rupture post AAD. The tunica adventitia of the expanded dissected aorta demonstrated high levels of interleukin-6 (IL-6) expression. Neutrophils were the major sources of IL-6, and CXCR2 neutralization significantly reduced local and systemic levels of IL-6. Furthermore, disruption of IL-6 effectively suppressed dilatation and rupture of the dissected aorta without any influence on the incidence of AAD and neutrophil mobilization. Conclusions: Adventitial CXCL1/granulocyte-colony stimulating factor expression in response to AAD triggers local neutrophil recruitment and activation. This leads to adventitial inflammation via IL-6 and results in aortic expansion and rupture.

Prepubertal Treatment with Angiotensin Receptor Blocker Causes Partial Attenuation of Hypertension and Renal Damage in Adult Dahl Salt-Sensitive Rats

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.

Functional characterization of podocan, a member of a new class in the small leucine-rich repeat protein family

An important component of the extracellular matrix is the group of non‐collagenous proteins belonging to the small leucine‐rich repeat (SLR) protein family. A new SLR protein, podocan, with structural characteristics different from the known classes of the SLR protein family has been identified recently from the kidney. In this study, we examined the functional characteristics of this SLR protein expressed in cultured cells. Podocan was clearly observed intracellularly and was also detectable in the supernatant. Treatment of the expressed protein with various glycoenzymes suggested that podocan is a glycoprotein containing N‐linked oligosaccharides but not a classical proteoglycan. Moreover, podocan was found to bind type 1 collagen. Cells transfected with podocan showed reductions in cell growth and migration, concomitant with increased p21 expression. Podocan mRNA was detected by reverse transcription polymerase chain reaction not only in the kidney, but also in other tissues including the heart and vascular smooth muscle cells, suggesting that podocan may have a potential role in growth regulation in cardiovascular tissues.

Extracellular Matrix Remodeling in Hypertension

Increases in arterial blood pressure cause cumulative changes in tissue structure and function, resulting ultimately in end-organ damage. One of the pathological hallmarks of hypertensive tissue injury is an increase in tissue fibrosis, which leads to reductions in tissue compliance and function. Fibrosis (or sclerosis) occurs as result of marked changes in the amount and composition of the extracellular matrix. This extracellular matrix is a complex mixture of structural proteins and glycoproteins, including collagens, fibronectins, and proteoglycans. Hypertension is known to be associated with increases in the synthesis of extracellular matrix proteins and changes in their degradation. These processes are mediated by several mediators, in particular the renin-angiotensin-aldosterone system. Since these changes play an important role in the formation of vascular sclerosis, cardiac dysfunction, and renal damage, understanding the mechanisms, and finding interventions to prevent or reverse these changes are clinically important. In this review we discuss the alterations in the extracellular matrix during hypertension, as well as the effects of antihypertensive agents in animal models and human patients.

Glomerular expression of biglycan and decorin and urinary levels of decorin in primary glomerular disease

AIMS Recent studies have suggested that small leucine-rich proteoglycans (SLRP) of the extracellular matrix play a major role in modulating the activity of growth factors and in regulating the deposition of collagens. In this study, the expression of the SLRPs biglycan and decorin in the glomeruli of patients with primary glomerular disease (minimal change disease, IgA nephropathy, and membranous nephropathy) and urine immunoreactive levels examined. METHODS Renal biopsy specimens were obtained from patients with minimal change disease, IgA nephropathy and membranous nephropathy. Immunohistochemical staining was performed on fresh-frozen samples using anti-biglycan and anti-decorin antibodies. Examination of urine proteoglycan excretion from a total of 26 patients and 8 normal volunteers was performed by indirect ELISA. RESULTS In normal kidney samples, biglycan and decorin expression was found predominantly in the intrarenal arteries and tubulointerstitium, with only minimal expression in the glomeruli. Glomerular expression of these proteoglycans in glomerular disease was unchanged in all of the 4 patients examined with minimal change disease. In the case of IgA nephropathy or membranous nephropathy, some of the patients showed minimally increased immunostaining of either biglycan or decorin, but there were no signs of simultaneous upregulation of both proteoglycans. To further examine the changes in proteoglycan expression, ELISA was performed on urine samples. Urine biglycan levels were below detection levels, but high values of urine decorin immunoreactivity were found in the patients with glomerular disease. A significant negative correlation was found between urine decorin and creatinine clearance. CONCLUSION These results suggest that distinct changes in the expression of the SLRPs biglycan and decorin may be seen in patients with primary glomerular disease. Moreover, the negative relationship between urine decorin levels and renal function supports the hypothesis that decorin may be involved in the pathophysiology of renal dysfunction in humans.

F4/80+ Macrophages Contribute to Clearance of Senescent Cells in the Mouse Postpartum Uterus

Cellular senescence, defined as an irreversible cell cycle arrest, exacerbates the tissue microenvironment. Our previous study demonstrated that mouse uterine senescent cells were physiologically increased according to gestational days and that their abnormal accumulation was linked to the onset of preterm delivery. We hypothesized that there is a mechanism for removal of senescent cells after parturition to maintain uterine function. In the current study, we noted abundant uterine senescent cells and their gradual disappearance in wild-type postpartum mice. F4/80+ macrophages were present specifically around the area rich in senescent cells. Depletion of macrophages in the postpartum mice using anti-F4/80 antibody enlarged the area of senescent cells in the uterus. We also found excessive uterine senescent cells and decreased second pregnancy success rate in a preterm birth model using uterine p53-deleted mice. Furthermore, a decrease in F4/80+ cells and an increase in CD11b+ cells with a senescence-associated inflammatory microenvironment were observed in the p53-deleted uterus, suggesting that uterine p53 deficiency affects distribution of the macrophage subpopulation, interferes with senescence clearance, and promotes senescence-induced inflammation. These findings indicate that the macrophage is a key player in the clearance of uterine senescent cells to maintain postpartum uterine function.

Investigation of metabolic factors associated with eGFR decline over 1 year in a Japanese population without CKD

Aim: Early intervention before the progression of chronic kidney disease (CKD) is essential to prevent end-stage renal disease (ESRD) and cardiovascular complications. This study evaluated the correlation between metabolic and lifestyle-related factors and the decline of estimated glomerular filtration rate (eGFR) over 1 year in a Japanese population without CKD. Methods: Subjects who received two consecutive annual health checkups from 2013 to 2015 were involved. Factors associated with eGFR decline were identified using multiple regression models. Results: A total of 2531 subjects aged 58.9 ± 11.7 years old were included in this study. Baseline levels of HDL-C and ApoA1 correlated with the eGFR decline over 1 year defined as eGFR reduction rate of 15% or more and/or eGFR at the next year < 60 ml/min/m2 (odds ratio (OR) 0.87 (per 10 mg/dl); 95% CI, 0.80–0.94; p = 0.0012, 0.90 (per 10 mg/dl); 0.86–0.96; p = 0.0004, respectively). A U-shaped relationship between the eGFR decline and HDL-C or ApoA1 levels was not observed in non-CKD population of this study. Metabolic syndrome was significantly associated with eGFR decline (OR 1.32; 1.04–1.67; p = 0.0205), although obesity-related factors did not show a significant correlation with eGFR decline over 1 year. Conclusion: Low HDL-C and ApoA1 levels significantly correlated with eGFR decline in a short period of 1 year. Metabolic syndrome also showed a significant association with eGFR decline. This study suggests the importance of hypertension and low HDL-C in the metabolic syndrome effect on eGFR decline rather than obesity in non-CKD population.