Transactivation domain of Krüppel-like factor 15 negatively regulates angiotensin II-induced adventitial inflammation and fibrosis.

Krüppel-like factor (KLF) 15 has emerged as a critical regulator of fibrosis in cardiovascular diseases. However, the precise role that KLF15 and its functional domain played in adventitial inflammation and fibrosis remains unclear. This study aims to investigate the role of the transactivation domain (TAD) of KLF15 in angiotensin II (Ang II)-induced adventitial pathologic changes. KLF15 expression was decreased in the vascular adventitia of Ang II-infused mice (1000 ng/kg/min, 14 d) and in adventitial fibroblasts (AFs) stimulated by Ang II (10-7 M). Adenovirus-mediated KLF15 overexpression normalized Ang II-induced vascular hypertrophy, increased collagen deposition, macrophage infiltration, and CCL2 and VCAM-1 expression. Interestingly, KLF15-ΔTAD (KLF15 with deletion of TAD at amino acids 132-152) overexpression showed no effect on the above pathologic changes. Similarly, perivascularly overexpression of KLF15 but not KLF15-ΔTAD in carotid arteries also attenuated Ang II-induced vascular inflammation and fibrosis. Furthermore, KLF15 overexpression after Ang II infusion rescued Ang II-induced vascular remodeling. CCL2 or VCAM-1-mediated monocyte and macrophage migration or adhesion to AFs in response to Ang II was negatively regulated by KLF15 through TAD. Ang II-enhanced Smad2/3 activation and adventitial migration, proliferation, and differentiation of AFs were suppressed by KLF15 but not KLF15-ΔTAD overexpression. Conversely, small interfering RNA knockdown of KLF15 aggravated Ang II-induced Smad2/3 activation and dysfunction of AFs. Luciferase, coimmunoprecipitation, and chromatin immunoprecipitation assay were used to demonstrate that interaction of KLF15 with Smad2/3 suppressed CCL2 expression through TAD. Mechanistically, activation of Ang II type 1 receptor/phospholipase Cγ 1/ERK1/2 signaling resulted in a decrease of KLF15 expression. In conclusion, these results demonstrate that KLF15 negatively regulates activation of AFs through TAD, which plays an important role in Ang II-induced adventitial inflammation and fibrosis.-Lu, Y.-Y., Li, X.-D., Zhou, H.-D., Shao, S., He, S., Hong, M.-N., Liu, J.-C., Xu, Y.-L., Wu, Y.-J., Zhu, D.-L., Wang, J.-G., Gao, P.-J. Transactivation domain of Krüppel-like factor 15 negatively regulates angiotensin II-induced adventitial inflammation and fibrosis.

Deficiency of Complement C3a and C5a Receptors Prevents Angiotensin II-Induced Hypertension via Regulatory T Cells.

RATIONALE: Inflammation and immunity play crucial roles in the development of hypertension. Complement activation-mediated innate immune response is involved in the regulation of hypertension and target-organ damage. However, whether complement-mediated T-cell functions could regulate blood pressure elevation in hypertension is still unclear. OBJECTIVE: We aim to determine whether C3aR (complement component 3a receptor) and C5aR (complement component 5a receptor) could regulate blood pressure via modulating regulatory T cells (Tregs). METHODS AND RESULTS: We showed that angiotensin II (Ang II)-induced hypertension resulted in an elevated expression of C3aR and C5aR in Foxp3 (forkhead box P3)+ Tregs. By using C3aR and C5aR DKO (double knockout) mice, we showed that C3aR and C5aR deficiency together strikingly decreased both systolic and diastolic blood pressure in response to Ang II compared with WT (wild type), single C3aR-deficient (C3aR-/-), or C5aR-deficient (C5aR-/-) mice. Flow cytometric analysis showed that Ang II-induced Treg reduction in the kidney and blood was also blocked in DKO mice. Histological analysis indicated that renal and vascular structure remodeling and damage after Ang II treatment were attenuated in DKO mice compared with WT mice. In vitro, Ang II was able to stimulate C3aR and C5aR expression in cultured CD4+CD25+ natural Tregs. CD3 and CD28 antibody stimuli downregulated Foxp3 expression in WT but not DKO Tregs. More important, depletion of Tregs with CD25 antibody abolished the protective effects against Ang II-induced hypertension and target-organ damage in DKO mice. Adoptive transfer of DKO Tregs showed much more profound protective effects against Ang II-induced hypertension than WT Treg transfer. Furthermore, we demonstrated that C5aR expression in Foxp3+ Tregs was higher in hypertensive patients compared with normotensive individuals. CONCLUSIONS: C3aR and C5aR DKO-mediated Treg function prevents Ang II-induced hypertension and target-organ damage. Targeting C3aR and C5aR in Tregs specifically may be an alternative novel approach for hypertension treatment.

Complement-mediated inhibition of adiponectin regulates perivascular inflammation and vascular injury in hypertension.

Perivascular adipose tissue (PVAT)-derived adiponectin (APN) is a secreted adipokine that protects against hypertension-related cardiovascular injury. However, the regulation of APN expression in hypertension remains to be explored. In this study, we demonstrated that down-regulation of APN was associated with complement activation in the PVAT of desoxycorticosterone acetate (DOCA)-salt hypertensive mice. Complement 3-deficient hypertensive mice were protected from ANP decrease in the PVAT. APN deficiency blockaded the protective effects of complement inhibition against hypertensive vascular injury. Mechanistically, complement 5a (C5a)-induced TNF-α secretion from macrophages is required for inhibiting APN expression in adipocytes. Macrophage depletion reversed C5a agonist peptide-induced TNF-α up-regulation and APN down-regulation in the PVAT of DOCA mice. Moreover, we detected increased macrophage infiltration and C5a expression associated with decreased APN expression in adipose tissue from patients with aldosterone-producing adenoma. These results identify a novel interaction between macrophages and adipocytes in the PVAT, where complement-mediated inhibition of APN acts as a potential risk factor for hypertensive vascular inflammation.-Ruan, C.-C., Ma, Y., Ge, Q., Li, Y., Zhu, L.-M., Zhang, Y., Kong, L.-R., Wu, Q-H., Li, F., Cheng, L., Zhao, A. Z., Zhu, D.-L., Gao, P.-J. Complement-mediated inhibition of adiponectin regulates perivascular inflammation and vascular injury in hypertension.

Complement-mediated macrophage polarization in perivascular adipose tissue contributes to vascular injury in deoxycorticosterone acetate-salt mice.

OBJECTIVE: We have previously shown an increased expression of complement 3 (C3) in the perivascular adipose tissue (PVAT) in the deoxycorticosterone acetate (DOCA)-salt hypertensive model. This study aims to examine the role and underlying mechanism of C3 in PVAT for understanding the pathogenesis of hypertensive vascular remodeling further. APPROACH AND RESULTS: The role of C3 in macrophage polarization was investigated using peritoneal macrophages from wild-type and C3-deficient (C3KO) mice because we found that C3 was primarily expressed in macrophages in PVAT of blood vessels from DOCA-salt mice, and results showed a decreased expression of M1 phenotypic marker in contrast to an increased level of M2 marker in the C3KO macrophages. Bone marrow transplantation studies further showed in vivo that DOCA-salt recipient mice had fewer M1 but more M2 macrophages in PVAT when the donor bone marrows were from C3KO compared with those from wild-type mice. Of note, this macrophage polarization shift was accompanied with an ameliorated vascular injury. Furthermore, we identified the complement 5a (C5a) as the major C3 activation product that was involved in macrophage polarization and DOCA-salt-induced vascular injury. Consistently, in vivo depletion of macrophages prevented the induction of C3 and C5a in PVAT, and ameliorated hypertensive vascular injury as well. CONCLUSIONS: The presence and activation of bone marrow-derived macrophages in PVAT are crucial for complement activation in hypertensive vascular inflammation, and C5a plays a critical role in DOCA-salt-induced vascular injury by stimulating macrophage polarization toward a proinflammatory M1 phenotype in PVAT.

Rare SNP rs12731181 in the miR-590-3p Target Site of the Prostaglandin F2α Receptor Gene Confers Risk for Essential Hypertension in the Han Chinese Population.

OBJECTIVE: To investigate whether rs12731181 (A→G) interrupted miR-590-3p-mediated suppression of the prostaglandin F2α receptor (FP) and whether it is associated with essential hypertension in the Chinese population. APPROACH AND RESULTS: We found that miR-590-3p regulates human FP gene expression by binding to its 3'-untranslated region. rs12731181 (A→G) altered the binding affinity between miR-590-3p and its FP 3'-untranslated region target, thus reducing the suppression of FP expression, which, in turn, enhanced FP receptor-mediated contractility of vascular smooth muscle cells. Overexpression of FP augmented vascular tone and elevated blood pressure in mice. An association study was performed to analyze the relationship between the FP gene and essential hypertension in the Han Chinese population. The results indicated that the rs12731181 G allele was associated with susceptibility to essential hypertension. Carriers of the AG genotype exhibited significantly higher blood pressure than those of the AA genotype. FP gene expression was significantly higher in human peripheral leukocytes from individuals with the AG genotype than that in leukocytes from individuals with the AA genotype. CONCLUSIONS: rs12731181 in the seed region of the miR-590-3p target site is associated with increased risk of essential hypertension and represents a new paradigm for FP involvement in blood pressure regulation.

Deletion of angiotensin-converting enzyme 2 exacerbates renal inflammation and injury in apolipoprotein E-deficient mice through modulation of the nephrin and TNF-alpha-TNFRSF1A signaling.

BACKGROUND: The renin-angiotensin system (RAS) has been implicated in atherosclerotic lesions and progression to chronic kidney diseases. We examined regulatory roles of angiotensin-converting enzyme 2 (ACE2) in the apolipoprotein E (ApoE) knockout (KO) kidneys. METHODS: The 3-month-old wild-type, ApoEKO, ACE2KO and ApoE/ACE2 double-KO (DKO) mice in a C57BL/6 background were used. The ApoEKO mice were randomized to daily deliver either Ang II (1.5 mg/kg) and/or human recombinant ACE2 (rhACE2; 2 mg/kg) for 2 weeks. We examined changes in pro-inflammatory cytokines, renal ultrastructure, and pathological signaling in mouse kidneys. RESULTS: Downregulation of ACE2 and nephrin levels was observed in ApoEKO kidneys. Genetic ACE2 deletion resulted in modest elevations in systolic blood pressure levels and Ang II type 1 receptor expression and reduced nephrin expression in kidneys of the ApoE/ACE2 DKO mice with a decrease in renal Ang-(1-7) levels. These changes were linked with marked increases in renal superoxide generation, NADPH oxidase (NOX) 4 and proinflammatory factors levels, including interleukin (IL)-1beta, IL-6, IL-17A, RANTES, ICAM-1, Tumor necrosis factor-alpha (TNF-alpha) and TNFRSF1A. Renal dysfunction and ultrastructure injury were aggravated in the ApoE/ACE2 DKO mice and Ang II-infused ApoEKO mice with increased plasma levels of creatinine, blood urea nitrogen and enhanced levels of Ang II in plasma and kidneys. The Ang II-mediated reductions of renal ACE2 and nephrin levels in ApoEKO mice were remarkably rescued by rhACE2 supplementation, along with augmentation of renal Ang-(1-7) levels. More importantly, rhACE2 treatment significantly reversed Ang II-induced renal inflammation, superoxide generation, kidney dysfunction and adverse renal injury in ApoEKO mice with suppression of the NOX4 and TNF-alpha-TNFRSF1A signaling. However, rhACE2 had no effect on renal NOX2 and TNFRSF1B expression and circulating lipid levels. CONCLUSIONS: ACE2 deficiency exacerbates kidney inflammation, oxidative stress and adverse renal injury in the ApoE-mutant mice through modulation of the nephrin, NOX4 and TNF-alpha-TNFRSF1A signaling. While rhACE2 supplementation alleviates inflammation, renal dysfunction and glomerulus injury in the ApoE-mutant mice associated with upregulations of Ang-(1-7) levels and nephrin expression and suppression of the TNF-alpha-TNFRSF1A signaling. Strategies aimed at enhancing the ACE2/Ang-(1-7) actions may have important therapeutic potential for atherosclerotic renal injury and kidney diseases.

The association of serum inflammatory biomarkers with chronic kidney disease in hypertensive patients.

A positive association between inflammation and chronic kidney disease (CKD) has been reported but the impact of hypertension on this relation remains unclear. The aim of this study is to investigate the association of various inflammation markers with risk of CKD in hypertensive patients. 387 hypertensive patients (mean age 55.5 years) were recruited. Serum matrix metalloproteinase-2 (MMP-2), matrix metalloproteinase-9 (MMP-9), tissue inhibitor of metalloproteinase-1(TIMP-1), high-sensitivity C-reactive protein (hsCRP) and osteopontin (OPN) were measured by ELISA. CKD was diagnosed either as evidence of kidney damage, including microalbuminuria, or by low glomerular filtration rate (GFR) (<60 mL/min/1.73 m(2)), which was estimated using the Modification of Diet in Renal Disease (MDRD) abbreviated equation. Compared with the reference groups (eGFR ≥ 60 mL/min/1.73 m(2)), the serum levels of TIMP-1, OPN, hsCRP were significantly higher, and the MMP-9/TIMP-1 ratio was lower in the risk group (eGFR < 60 mL/min/1.73 m(2)). Multiple logistic regression analysis showed that TIMP-1, MMP-9/TIMP-1 ratio, OPN and hsCRP were associated with low GFR separately after adjustment, whereas MMP-9/TIMP-1 ratio, OPN and hsCRP were associated with microalbuminuria. The significant association of MMP-9/TIMP-1 ratio and OPN with low GFR and microalbuminuria persisted after additional adjustment for other studied inflammatory biomarkers. Our data suggest that inflammation is strongly and independently associated with renal damage in hypertensive patients. MMP-9/TIMP-1 ratio and OPN may serve as novel risk factors and therapeutic targets for the treatment of CKD in hypertensive patients.

Cardiac protective effects of irbesartan via the PPAR-gamma signaling pathway in angiotensin-converting enzyme 2-deficient mice.

BACKGROUND: Angiotensin-converting enzyme 2 (ACE2), a monocarboxypeptidase which metabolizes angiotensin II (Ang II) to generate Ang-(1-7), has been shown to prevent cardiac hypertrophy and injury but the mechanism remains elusive. Irbesartan has the dual actions of angiotensin receptor blockade and peroxisome proliferator-activated receptor-γ (PPARγ) activation. We hypothesized that irbesartan would exert its protective effects on ACE2 deficiency-mediated myocardial fibrosis and cardiac injury via the PPARγ signaling. METHODS: 10-week-old ACE2 knockout (ACE2KO; Ace2(-/y)) mice received daily with irbesartan (50 mg/kg) or saline for 2 weeks. The wild-type mice (Ace2(+/y)) were used to the normal controls. We examined changes in myocardial ultrastructure, fibrosis-related genes and pathological signaling by real-time PCR gene array, Western blotting, Masson trichrome staining and transmission electron microscope analyses, respectively. RESULTS: Compared with the Ace2(+/y) mice, cardiac expression of PPARα and PPARγ were reduced in Ace2(-/y) mice and the myocardial collagen volume fraction (CVF) and expression of fibrosis-related genes were increased, including transforming growth factor-ß1 (TGFß1), connective tissue growth factor (CTGF), collagen I and collagen III. Moreover, ACE2 deficiency triggered cardiac hypertrophy, increased myocardial fibrosis and adverse ultrastructure injury in ACE2KO hearts with higher levels of atrial natriuretic factor (ANF) and phosphorylated extracellular signal-regulated kinase 1/2 (ERK1/2), without affecting cardiac systolic function. Intriguingly, treatment with irbesartan significantly reversed ACE2 deficiency-mediated pathological hypertrophy and myocardial fibrosis in Ace2(-/y) mice linked with enhancement of plasma Ang-(1-7) level and downregulation of AT1 receptor in heart. Consistent with attenuation of myocardial fibrosis and ultrastructure injury, the myocardial CVF and levels of ANF, TGFß1, CTGF, collagen I, collagen III and phosphorylated ERK1/2 were lower, and expression of PPARγ was higher in ACE2KO mice in response to irbesartan treatment, without affecting cardiac expression of PPARα, PPARδ, ß-myosin heavy chain, TGFß2 and fibronectin. CONCLUSIONS: We conclude that irbesartan prevents ACE2 deficiency-mediated pathological hypertrophy and myocardial fibrosis in ACE2 mutant mice via activation of the PPARγ signaling and suppression of the TGFß-CTGF-ERK signaling, resulting in attenuation of myocardial injury. Drugs targeting ACE2 and PPARγ represent potential candidates to prevent and treat myocardial injury and related cardiac disorders.

Vascular endothelial growth factor-induced osteopontin expression mediates vascular inflammation and neointima formation via Flt-1 in adventitial fibroblasts.

OBJECTIVE: Adventitia acts as an active participant in vascular inflammation but the precise mechanism underlying adventitia-mediated vascular inflammation is not fully understood. In this study, we sought to determine whether vascular endothelial growth factor (VEGF) regulates osteopontin (OPN) expression through Flt-1 in adventitial fibroblasts (AFs) to mediate vascular inflammation and neointima formation. METHODS AND RESULTS: In primary cultured AFs, VEGF increased intracellular and secreted OPN expression in a time- and dose-dependent manner, which was effectively suppressed by a specific anti-Flt-1 hexapeptide. Interestingly, VEGF treatment of AFs enhanced the capability of AF-conditioned medium to stimulate macrophages chemotaxis, and this effect was attenuated after blockade of OPN from AF-conditioned medium. Furthermore, perivascular delivery of anti-Flt-1 peptide preferentially concentrated in the adventitia resulted in a decrease of neointima formation after balloon injury in carotid arteries. The inhibition of neointima formation was preceded by significant reduction of VEGF and OPN expression with concurrent macrophage infiltration into adventitia after injury. Activation of extracellular signal-regulated kinase 1/2 pathway was involved in OPN upregulation and macrophage chemotaxis. CONCLUSIONS: These results demonstrate that VEGF/Flt-1 signaling plays a significant role in vascular inflammation and neointima formation by regulating OPN expression in AFs and provide insight into Flt-1 as a potential therapeutic target for vascular diseases.

Loss of angiotensin-converting enzyme 2 exacerbates myocardial injury via activation of the CTGF-fractalkine signaling pathway.

BACKGROUND: Angiotensin-converting enzyme 2 (ACE2) has been implicated in human heart failure, but the mechanism remains elusive. We hypothesized that ACE2 deficiency would exacerbate angiotensin (Ang) II-mediated myocardial injury. METHODS AND RESULTS: 10-week-old ACE2 knockout (ACE2KO) and wild-type mice received by mini-osmotic pump either AngII (1.5 mg·kg(-1)·day(-1)) or saline for 2 weeks. ACE2 deficiency triggered greater increases in the expression of connective tissue growth factor (CTGF), fractalkine (FKN) and phosphorylated ERK1/2 in AngII-treated ACE2KO hearts. These changes were associated with greater activation of matrix metalloproteinase (MMP) 2, MMP9 and MT1-MMP and exacerbation of myocardial injury and dysfunction. In cultured cardiofibroblasts, exposure to AngII (100 nmol/L) for 30 min resulted in marked increases in superoxide production and expression of CTGF, FKN and phosphorylated ERK1/2, which were strikingly prevented by recombinant human ACE2 (rhACE2; 1mg/ml) and the CTGF-neutralizing antibody (5 µg/ml), but were aggravated by ACE2 inhibitor DX600 (0.5 µmol/L). These protective effects of rhACE2 were eradicated by the Ang-(1-7) antagonist A779 (1 µmol/L). More intriguingly, rhACE2 treatment significantly abolished AngII-mediated increases in MMP2, MMP9 and MT1-MMP in cardiofibroblasts. CONCLUSIONS: Loss of ACE2 exacerbates AngII-mediated inflammation, myocardial injury and dysfunction in ACE2-deficient hearts via activation of the CTGF-FKN-ERK and MMP signaling. ACE2 gene may represent a potential candidate to prevent and treat myocardial injury and heart diseases.

Apocynin attenuates oxidative stress and cardiac fibrosis in angiotensin II-induced cardiac diastolic dysfunction in mice.

AIM: To investigate whether apocynin, a NADPH oxidase inhibitor, produced cardioproteictive effects in Ang II-induced hypertensive mice, and to elucidate the underlying mechanisms. METHODS: C57BL/6 mice were subcutaneously infused Ang II for 4 weeks to mimic cardiac remodeling and fibrosis. Concomitantly the mice were administered apocynin (100 mg·kg(-1)·d(-1)) or/and the aldosterone receptor blocker eplerenone (200 mg·kg(-1)·d(-1)) via gavage for 4 weeks. Systolic blood pressure (SBP) and heart rate were measured, and transthoracic echocardiography was performed. For in vitro study, cardiac fibroblasts were treated with Ang II (10(-7) mol/L) in the presence of apocynin (10(-5) mol/L) or/and eplerenone (10(-5) mol/L). Immunohistochemistry and Western blotting were used to quantify the expression levels of NADPH oxidase and osteopontin (OPN) proteins in the cells. RESULTS: Both apocynin and eplerenone significantly decreased SBP, and markedly improved diastolic dysfunction in Ang II-induced hypertensive mice, accompanied with ameliorated oxidative stress and cardiac fibrosis. In the Ang II-treated cardiac fibroblasts, the expression levels of NOX4 and OPN proteins were markedly upregulated. Both Apocynin and eplerenone significantly suppressed the increased expression levels of NOX4 and OPN proteins in the Ang II-treated cells. In all the experiments, apocynin and eplerenone produced comparable effects. Co-administration of the two agents did not produce synergic effects. CONCLUSION: Apocynin produces cardioproteictive effects comparable to those of eplerenone. The beneficial effects of apocynin on myocardial oxidative stress and cardiac fibrosis might be mediated partly through a pathway involving NADPH oxidase and OPN.

Perivascular adipose tissue-derived complement 3 is required for adventitial fibroblast functions and adventitial remodeling in deoxycorticosterone acetate-salt hypertensive rats.

OBJECTIVE: To examine the role of perivascular adipose tissue (PVAT)-derived factors in the regulation of adventitial fibroblast (AF) function in vitro and in vivo. METHODS AND RESULTS: PVAT is an active component of blood vessels. Bioactive substances released from PVAT play regulatory roles in vascular function. However, their effects on vascular AFs remain unclear. PVAT-conditioned medium stimulated AF migration using a transwell technique, and differentiation was evaluated by α-smooth muscle-actin induction. We identified the secretome of PVAT by liquid chromatography-tandem mass spectrometry. One of the major secretory proteins in PVAT is complement 3 (C3). The C3 antagonist and neutralizing antibody attenuated PVAT-conditioned medium-induced AF migration and differentiation. Similar to PVAT-conditioned medium, C3 recombinant protein stimulated AF migration and differentiation. We demonstrated that the effects of PVAT-derived C3 were mediated by the c-Jun N-terminal kinase pathway. Moreover, we found morphological changes in perivascular adipocytes and increased expression of C3 in PVAT that was tightly associated with adventitial thickening and myofibroblast clustering around PVAT in deoxycorticosterone acetate-salt hypertensive rats. CONCLUSIONS: PVAT-derived C3 stimulated AF migration and differentiation via the c-Jun N-terminal kinase pathway. PVAT-derived C3 may contribute to adventitial remodeling in a deoxycorticosterone acetate-salt hypertensive model.

Effect of an acute mechanical stimulus on aortic structure in the transverse aortic constriction mouse model.

1. Vascular remodelling is an adaptive response to various stimuli, including mechanical forces, inflammatory cytokines and hormones. In the present study, we investigated histological modification of the aorta and the expression of key proteins participating in vascular remodelling under an acute mechanical stimulus using a transverse aortic constriction (TAC) mouse model. 2. The TAC was performed in male C57BL/6 mice aged 10-12 weeks. A Millar conductance catheter was used to measure cardiac haemodynamic parameters 3 and 14 days after TAC. Aortic structural variations were observed by haematoxylin and eosin, Sirius red and Weigert's elastin staining. Protein levels of Type I collagen, F4/80, α-smooth muscle actin (SMA) and SM22α were analysed by immunohistochemistry. 3. Three days after TAC, the medial area proximal to the aortic band (PA-B) was increased, whereas the area distal to the aortic band (DA-B) was unchanged. There was no difference in luminal area between TAC and sham groups. The adventitia displayed the most significant difference 14 days after TAC: adventitial hyperplasia was abundant and collagen was upregulated in the adventitia of the PA-B with a considerable increase in α-SMA and SM22α. Macrophages accumulated in the adventitia of the PA-B 3 days after TAC and infiltrated into the media and intima of the PA-B 14 days after TAC. 4. In conclusion, the aortic structure undergoes considerable remodelling following an acute mechanical stimulus in the TAC model, mainly in the adventitia. Upregulation of α-SMA and extracellular matrix components accompanied by macrophage infiltration may contribute to adventitial modification in the TAC mouse model.

Association of stroke with ambulatory arterial stiffness index (AASI) in hypertensive patients.

The ambulatory arterial stiffness index (AASI) predicted stroke in hypertensive patients and in the general populations. However, no similar data was available in Chinese. In the present study, we sought confirmation that Chinese hypertensive patients with a history of stroke would have an elevated AASI. We retrospectively analyzed the data of 156 hypertensive outpatients (60.9 % men; mean age, 61.5 years) and 582 inpatients (63.6 % men; 58.6 years) of the Hypertension Department at Ruijin Hospital in Shanghai, China. The AASI was calculated as 1 - the regression slope of diastolic blood pressure (DBP) on systolic blood pressure (SBP) in individual 24-h ambulatory recordings. With adjustment applied for sex, age, body mass index (BMI), the 24-h mean arterial pressure, and other cardiovascular risk factors, AASI was higher in patients with a history of stroke than in patients without stroke in both outpatient (0.51 ± 0.02 vs. 0.47 ± 0.01; P = 0.050) and inpatient (0.46 ± 0.01 vs. 0.44 ± 0.01; P = 0.031) cohorts. The odds ratio (OR) for a history of stroke associated with a 1-SD increase in AASI was 1.63 (95% confidence interval (CI), 1.01-2.62; P = 0.046) in outpatients, 1.32 (1.01-1.74; P = 0.046) in inpatients, and 1.30 (1.05-1.62; P = 0.018) in two patient cohorts combined (n = 738) after multivariate adjustment including the night-to-day ratio of SBP. Our findings suggest that Chinese hypertensive patients with a history of stroke, compared to those without such history, have stiffer arteries, as exemplified by a higher AASI.

MicroRNA-155 regulates angiotensin II type 1 receptor expression and phenotypic differentiation in vascular adventitial fibroblasts.

MicroRNAs (miRNAs), which are genomically encoded small RNAs, negatively regulate target gene expression at the post-transcriptional level. Our recent study indicated that microRNA-155 (miR-155) might be negatively correlated with blood pressure, and it has been suggested that miR-155-mediated target genes could be involved in the cardiovascular diseases. Bioinformatic analyses predict that angiotensin II type 1 receptor (AT(1)R) is a miR-155 target gene. The present study investigated the potential role of miR-155 in regulating AT(1)R expression and phenotypic differentiation in rat aortic adventitial fibroblasts (AFs). Luciferase assay demonstrated that miR-155 suppressed AT(1)R 3'-UTR reporter construct activity. miR-155 overexpression in AFs did not reduce target mRNA levels, but significantly reduced target protein expression. In addition, AFs transfected with pSUPER/miR-155 exhibited reduced Ang II-induced ERK1/2 activation. miR-155 overexpression in cells attenuated Ang II-induced α-smooth muscle actin (α-SMA, produces myofibroblast) expression, but did not transform growth factor beta-1 (TGF-ß1). This study demonstrated that miR-155 could have an important role in regulating adventitial fibroblast differentiation and contribute to suppression of AT(1)R expression.

Effect of hirulog-like peptide on balloon catheter injury-induced neointimal formation in femoral arteries of minipigs and relationship with inflammatory mediators.

Vascular intervention-induced neointimal formation is a major drawback for managing atherosclerotic cardiovascular diseases using invasive vascular procedures. Our previous studies demonstrated that hirulog-like peptide (HLP) reduced balloon catheter dilation-induced neointimal formation or restenosis in carotid arteries of rats or atherosclerotic rabbits with less interruption in coagulation or bleeding than heparin or hirulog-1. The present study examined the effect of HLP on balloon catheter injury-induced neointimal formation in femoral arteries of minipigs. Intravenous infusion of HLP (1.6 mg/kg/h for 4 h started 0.5 h before the intervention) or unfractured heparin (50 U/kg/h for 4 h) significantly reduced neointimal formation in femoral arteries 4 weeks after intervention compared with the vehicle. Heparin, but not HLP, significantly prolonged activated partial thromboplastin time. HLP or heparin significantly reduced vascular intervention-induced increases in C-reactive protein, P-selectin and interleukin-6 in serum. HLP, but not heparin, normalized vascular injury-induced increase in P-selectin in platelets. The results of the present study suggest that HLP is an effective agent for preventing balloon catheter injury-induced neointimal formation in femoral arteries of minipigs. The beneficial effects of HLP on vascular injury-induced neointimal formation may partially result from its inhibition on inflammatory mediators.

The PDE1A-PKCalpha signaling pathway is involved in the upregulation of alpha-smooth muscle actin by TGF-beta1 in adventitial fibroblasts.

BACKGROUND: Increasing evidence has suggested that differentiation of adventitial fibroblasts (AFs) to myofibroblasts plays an important role in arterial remodeling. The molecular mechanisms by which myofibroblast formation is regulated still remain largely unknown. This study aimed to evaluate the role of cyclic nucleotide phosphodiesterase 1A (PDE1A) in the formation of adventitial myofibroblasts induced by transforming growth factor (TGF)-beta(1). METHODS AND RESULTS: AFs were cultured by the explant method. Western blot and immunocytochemistry were applied for alpha-smooth muscle actin (SMA) or protein kinase C (PKC) alpha protein analysis. Results showed that TGF-beta(1) upregulated PDE1A protein expression in rat aortic AFs and pharmacological inhibition of PDE1A blocked TGF-beta(1)-induced alpha-SMA expression, a marker of myofibroblast formation, suggesting that the upregulation of PDE1A may mediate TGF-beta(1)-induced AF transformation. Moreover, calphostin C (a PKC inhibitor) inhibited TGF-beta(1)-induced alpha-SMA expression, whereas phorbol-12-myristate-13-acetate (a PKC activator) induced it. Finally, the upregulation of PKCalpha expression by TGF-beta(1) was also inhibited by PDE1A inhibition. CONCLUSIONS: Taken together, our data suggest that TGFbeta(1) induces alpha-SMA expression and myofibroblast formation via a PDE1A-PKCalpha-dependent mechanism. Our study thus unveils a novel signaling mechanism underlying TGF-beta(1)-induced adventitial myofibroblast formation.

Different biomechanical properties of medial and adventitial layers of thoracic aorta in Wistar-Kyoto and spontaneously hypertensive rats.

AIM: To evaluate the biomechanical properties of thoracic aorta with or without adventitia, and to determine whether there are corresponding changes with hypertension. METHODS: Normotensive Wistar-Kyoto (WKY) rats and spontaneously hypertensive rats (SHR) at the age of 16 and 32 weeks were used. Thoracic aortic adventitial layer was mechanically separated from thoracic aorta and the adventitia-denuded artery ring was viewed as thoracic media. A load-strain curve was obtained by stretching the ring-shaped intact thoracic aorta or thoracic media with a tensile testing machine. Then, the slope of the load-stain curve at 30%-40% strains was viewed as the elastic stiffness at physiological load, whereas the slope near the breaking point was calculated as maximum stiffness. The maximum load is the load at the breaking point. RESULTS: There was no significant difference in elastic stiffness and maximum stiffness of intact thoracic aorta between SHR and age-matched WKY. The elastic stiffness of intact thoracic aorta showed no significant difference from that of thoracic media in WKY and SHR at both ages. In contrast, both maximum stiffness and maximum load were reduced in thoracic media compared with intact thoracic aorta in SHR and WKY at both ages. CONCLUSION: These results indicated that vascular adventitia contributes to maximum stiffness, but not elastic stiffness in both SHR and WKY.

Dynamic expression of proteins associated with adventitial remodeling in adventitial fibroblasts from spontaneously hypertensive rats.

AIM: To identify proteins that could potentially be involved in adventitial remodeling in vascular adventitial fibroblasts (AFs) from spontaneously hypertensive rats (SHR). METHODS: AFs were isolated from thoracic aortas of 4-, 8-, 16-, and 24-week-old male SHR and Wistar-Kyoto (WKY) rats and cultured to passage 4. Proteomic differential expression profiles between SHR-AFs and WKY-AFs were investigated using 2-D electrophoresis (2-DE), whereas gel image analysis was processed using Image Master 2D Platinum. Protein spots were identified using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Expression levels of annexin A1 in AFs and aortas from SHR and WKY rats were detected with Western blotting and immunofluorescence techniques. RESULTS: In 4-, 8-, 16-, and 24-week-old SHR-AFs, 49, 59, 54, and 69 protein spots were found to have significant differences from the age-matched WKY-AFs. Fourteen spots with the same changes in patterns were analyzed in 4-, 8-, 16-, and 24-week-old SHR-AFs with mass spectrometry. Except for cytoskeleton proteins such as tubulin beta 5, it was found that annexin A1, translation elongation factor Tu, endoplasmic reticulum protein 29 and calcium-binding protein 1 were expressed in vascular AFs and their levels changed significantly in SHR-AFs compared with those in WKY-AFs. A decrease in annexin A1 in SHR-AFs was confirmed with Western blotting and immunofluorescence staining at the cell and tissue levels. CONCLUSION: The application of proteomic techniques revealed a number of novel proteins involved in adventitial remodeling of AFs from SHR, which provide new mechanisms responsible for the occurrence and development of hypertension and potential targets for influencing vascular remodeling in hypertension.

Adventitial fibroblast-derived vascular endothelial growth factor promotes vasa vasorum-associated neointima formation and macrophage recruitment.

AIMS: Adventitial vasa vasorum provides oxygen and nourishment to the vascular wall, but whether it regulates vascular disease remains unclear. We have previously shown that an increased expression of VEGF (vascular endothelial growth factor) is associated with macrophage infiltration. This study aims to determine whether adventitial fibroblast (AF)-derived VEGF increases the number of vasa vasorum contributing to neointima formation through macrophage recruitment. METHODS AND RESULTS: In rat balloon injury model, vasa vasorum count was increased particularly in the adventitia accompanied by cell proliferation and VEGF expression. Both endogenous and PKH26-labelled exogenous macrophages were mainly distributed in adventitia around vasa vasorum. Interestingly, perivascular delivery of Ranibizumab preferentially concentrated in adventitia resulted in a decrease of neointima formation with concurrent reduction of vasa vasorum count and macrophage infiltration. AFs with adenovirus-mediated VEGF over-expression delivered to the adventitia significantly enhanced these pathological changes after injury. In Tie2-cre/Rosa-LoxP-RFP mice, endothelial cells were increased in the adventitia after wire injury. By using multiphoton laser scanning microscopy, macrophage rolling, adhesion and transmigration were observed in vasa vasorum. Moreover, adoptive transfer of macrophages accelerated injury-induced neointima formation. VEGF-neutralizing antibody administration also attenuated wire injury-induced neointima formation and macrophage infiltration. In primary cultured AFs, exogenous VEGF increased VEGF expression and secretion in a time- and dose-dependent manner. AF-conditioned medium promoted endothelial cell angiogenesis, vascular cell adhesion molecule-1 expression and macrophage adhesion was blocked by VEGF-neutralizing antibody and VEGFR2 inhibitor ZM323881, which also inhibited activation of VEGFR2/ERK1/2 pathway. CONCLUSION: These results demonstrate that AF-derived VEGF plays a significant role in the increase of vasa vasorum count which is involved in macrophage recruitment and neointima formation.