Vascular Adventitial Fibroblasts Research Articles

Decreased expression of P54nrb/NonO correlates with collagen deposition and fibrosis in human aortic dissection

Aortic dissection (AD) is characterized by changes in the extracellular matrix, including fibrosis with collagen production. P54(nrb) /NonO is known to be involved in collagen formation. In this study, we examined whether AD is associated with abnormal P54(nrb) /NonO expression. Aortic specimens and serum were obtained from 10 patients with AD and 10 controls. In-vitro cultures of vascular smooth muscle cells (VSMCs) and adventitial fibroblasts (AFs) were obtained from organ donors. P54(nrb) /NonO protein and mRNA levels were determined by Western blot, immunohistochemistry and quantitative real-time reverse transcription-polymerase chain reaction (quantitative real-time RT-PCR). To evaluate collagen expression, we stained tissue sections with Masson's trichrome. Serum concentration of TNF-α was determined by enzyme-linked immunosorbent assay (ELISA). Aortic P54(nrb) /NonO protein and mRNA were decreased in AD patients, compared with controls. Decreased P54(nrb) /NonO mRNA correlated significantly with increased collagen deposition and fibrosis in AD aortas. In VSMCs and AFs from normal human aortas, P54(nrb) /NonO was expressed strongly and localized to the nucleus. Patients with AD exhibited significantly decreased expression of P54(nrb) /NonO. The significant correlation between P54(nrb) /NonO and collagen may point to novel thinking about collagen metabolism research in AD aorta.

Inhibition of phosphorylated-STAT1 nuclear translocation and antiviral protein expression in human brain vascular adventitial fibroblasts infected with varicella-zoster virus.

In varicella-zoster virus (VZV)-infected primary human brain vascular adventitial fibroblasts (BRAFs), levels of beta interferon (IFN-β,) STAT1, and STAT2 transcripts as well as STAT1 and STAT2 protein were decreased. IFN-α transcript levels were increased but not secreted IFN-α protein levels. Compared to IFN-α-treated control results, in VZV-infected BRAFs, phosphorylated STAT1 did not translocate to the nucleus, resulting in impaired downstream expression of interferon-inducible antiviral Mx1. Overall, VZV interference with the type I interferon pathway may promote virus persistence in cerebral arteries.

The interaction of transient receptor potential melastatin 7 with macrophages promotes vascular adventitial remodeling in transverse aortic constriction rats

Transient receptor potential melastatin 7 (TRPM7), a novel channel kinase, has been recently identified in the vasculature. However, its regulation and function in vascular diseases remain poorly understood. To address this lack of knowledge, we sought to examine whether TRPM7 can mediate the vascular remodeling process induced by pressure overload in the right common carotid artery proximal to the band (RCCA-B) in male Sprague-Dawley rats with transverse aortic constriction (TAC). The contribution of TRPM7 to amplified vascular remodeling after TAC was tested using morphometric and western blot analyses. Pressure overload-induced vascular wall thickening, especially in the adventitia, was readily detected in RCCA-B. The TRPM7 level was increased with a simultaneous accumulation of macrophages in the adventitia of RCCA-B, whereas the anti-inflammatory molecule annexin-1, a TRPM7 downstream target, was decreased. After the addition of the TRPM7 inhibitor 2-aminoethoxydiphenyl borate (2-APB), significant reductions in macrophage accumulation as well as the expression of monocyte chemotactic protein-1, SM-22-α and collagen I were observed, whereas annexin-1 was rescued. Finally, in cultured vascular adventitial fibroblasts treated with macrophage-conditioned medium, there were marked increases in the expression of TRPM7 and SM-22-α with a concurrent reduction in annexin-1 expression; these effects were largely prevented by treatment with 2-APB and specific anti-TRPM7 small interfering RNA. Our findings provide the first demonstration of the potential regulatory roles of TRPM7 in the vascular inflammation, pressure overload-mediated vascular adventitial collagen accumulation and cell phenotypic transformation in TAC rats. The targeting of TRPM7 has potential therapeutic importance for vascular diseases.

SIRT1 inhibits TNF-α-induced apoptosis of vascular adventitial fibroblasts partly through the deacetylation of FoxO1

Sirtuin 1 (SIRT1), a NAD(+)-dependent class III histone deacetylase, participates in regulating cellular apoptosis, senescence and metabolism by deacetylating histones and multiple transcription factors. In this study, we aimed to determine the effect of SIRT1 on the apoptosis of vascular adventitial fibroblasts (VAFs) and related signaling pathways. SIRT1 was found in the nucleus of VAFs and translocated into the cytoplasm in response to tumor necrosis factor-α (TNF-α). Moreover, SIRT1 protein expression was reduced in VAFs stimulated with TNF-α. In addition, TNF-α increased the apoptosis of VAFs. Activation of SIRT1 by resveratrol (RSV) or overexpression of SIRT1 attenuated TNF-α-induced VAF apoptosis by decreasing the percentage of apoptotic cells and cleaved caspase-3 protein expression and increasing the Bcl-2/Bax ratio. In contrast, inhibition of SIRT1 by sirtinol/nicotinamide or knockdown of SIRT1 enhanced apoptosis of VAFs. On the other hand, knockdown of FoxO1 reduced TNF-α-induced VAF apoptosis. SIRT1 interacted with FoxO1 in VAFs by the co-immunoprecipitation assay. Further study showed that RSV or SIRT1 overexpression decreased acetylated-FoxO1 (Ac-FoxO1) protein expression in VAFs stimulated with TNF-α. Knockdown of SIRT1 resulted in an increase in Ac-FoxO1 protein expression. Taken together, these findings indicate that SIRT1 inhibits the apoptosis of VAFs, whereas FoxO1 promotes VAF apoptosis. Furthermore, the inhibitory effect of SIRT1 on VAF apoptosis is partly mediated by the deacetylation of FoxO1.

The NADPH Oxidase Family and its Inhibitors

The classical nicotinamide adenine dinucleotide phosphate (NADPH) oxidase was originally detected in neutrophils as a multicomponent enzyme that catalyzes the generation of superoxide from oxygen and the reduced form of NADPH. This enzyme is composed of two membrane-bound subunits (p22phox and gp91phox), three cytosolic subunits (p67phox, p47phox, and p40phox) and a small G-protein Rac (Rac1 and Rac2). Recently, it has been demonstrated that there are several isoforms of nonphagocytic NADPH oxidase. Endothelial cells, vascular smooth muscle cells or adventitial fibroblasts possess multiple isoforms of this enzyme. The new homologs, along with gp91phox are now designated the Nox family of NADPH oxidases and are key sources of reactive oxygen species in the vasculature. Reactive oxygen species play a significant role in regulating endothelial function and vascular tone. However, besides the participation in the processes of physiological cell, these enzymes can also be the perpetrator of oxidative stress that causes endothelial dysfunction. This review summarizes the current state of knowledge of the structure and functions of NADPH oxidase and NADPH oxidase inhibitors in the treatment of disorders with endothelial damage.

Abstract 420: Synthesis and Targeted Spatiotemporal Delivery of Peptide Amphiphile to the Vasculature

Introduction: The aim of this study is to develop and evaluate a novel systemically delivered targeted therapy that will prevent restenosis after all cardiovascular interventions. To accomplish this aim, our goal is to synthesize a self-assembled peptide amphiphile (PA) targeted to a unique collagen-binding sequence, and demonstrate binding to the site of vascular intervention. Our hypothesis is that our collagen binding PA will bind to the site of vascular injury. Methods: PA was synthesized using solid-phase peptide synthesis and purified by reversed-phase HPLC. The collagen-binding sequence and a fluorescent tag (AlexaFluor546) was incorporated into the PA. Vascular smooth muscle cells (VSMC) and adventitial fibroblasts (AF) were exposed to the PA (0.25-1.0 mM) in culture and metabolic activity was assessed with the MTT assay. Male Sprague Dawley rats underwent the carotid artery injury model followed by intra-arterial injection of the PA (5mg) for 10min. Carotid arteries and arterial cross-sections were imaged for fluorescence using excitation and emission wavelengths of 535 nm and 580 nm, resp. Results: VSMC and AF exposed to the targeted PA for 24hrs showed similar metabolic activity compared to controls except at the highest concentration (1mM) at which a 50% decrease was detected (P<0.05). Carotid arteries injured and exposed to intra-arterial PA followed by intraluminal flushing with PBS exhibited positive binding of the PA to the site of injury with 5-fold greater fluorescent intensity compared to background levels. Similarly, carotid arteries injured and exposed to PA in a similar manner followed by re-establishment of blood flow for 20min exhibited 3-fold greater fluorescent intensity than background levels. Arterial cross sections demonstrated binding of the PA to the intima. No significant fluorescent signal was detected in the uninjured arteries. Conclusion: These data demonstrate that our novel targeted PA is biocompatible with vascular cells in vitro. Furthermore, we successfully demonstrated specific binding of our injectable targeted PA to the site of injury. This research serves as the foundation upon which a targeted drug-eluting therapy will be designed and evaluated to prevent restenosis following vascular interventions.

Single-walled carbon nanotubes promote rat vascular adventitial fibroblasts to transform into myofibroblasts by SM22-α expression

The aim of this study was to explore whether single-wall carbon nanotubes (SWCNTs) can be used as artery tissue-engineering materials by promoting vascular adventitial fibroblasts (VAFs) to transform into myofibroblasts (MFs) and to find the signal pathway involved in this process. VAFs were primary cultured and incubated with various doses of SWCNTs suspension (0, 0.8, 3.2, 12.5, 50, and 200 μg/mL). In the present study, we used three methods (MTT, WST-1, and WST-8) at the same time to detect the cell viability and immunofluorescence probe technology to investigate the effects of oxidative injury after VAFs incubated with SWCNTs. Immunocytochemical staining was used to detect SM22-α expression to confirm whether VAFs transformed into MFs. The protein levels were detected by western blotting. The results of immunocytochemical staining showed that SM22-α was expressed after incubation with 50 μg/mL SWCNTs for 96 hours, but with oxidative damage. The mRNA and protein levels of SM22-α, C-Jun N-terminal kinase, TGF-β1, and TGF-β receptor II in VAFs increased with the dose of SWCNTs. The expression of the p-Smad2/3 protein was upregulated while the Smad7 protein was significantly down-regulated. Smad4 was translocated to the nucleus to regulate SM22-α gene expression. In conclusion, SWCNTs promoted VAFs to transform into MFs with SM22-α expression by the C-Jun N-terminal kinase/Smads signal pathway at the early stage (48 hours) but weakened quickly. SWCNTs also promoted the transformation by the TGF-βl/Smads signal pathway at the advanced stage in a persistent manner. These results indicate that SWCNTs can possibly be used as artery tissue-engineering materials.

Cardiovascular-a new novel source of catestatin and its precursor chromogranin A

ObjectiveTo investigate the expression and distribution of catestatin and its precursor phromogranin A in tissue and cardiovascular cell levels.MethodExtract RNA from Wistar rat heart tissue, vascular tissue, cardiomyocytes, cardiac fibroblasts,...

Expression and function of osteopontin in vascular adventitial fibroblasts and pathological vascular remodeling

Objectives: Osteopontin (OPN) is known to play important role in various diseases including vascular disorders. However, little is known about its expression and function in vascular adventitial fibroblasts. Adventitial fibroblasts have been shown to play a key role in pathological vascular remodeling associating with various vascular disorders. In this study, activation of OPN and its biological functions was investigated. Methods and results: Angiotensin II and aldosterone increased OPN expression in adventitial fibroblasts in a time- and concentration-dependent manner.

Metformin may inhibit cardiovascular remodeling through suppressing urotensin II and angiotensin II system

BackgroundCardiovascular complications of diabetes always affect the prognosis of patients. Cardiovascular remodeling characterised by proliferation and collagen synthesis of cardiovascular cells is the key stones of cardiovascular complications. Urotensin II...

Angiotensin II downregulates catalase expression and activity in vascular adventitial fibroblasts through an AT1R/ERK1/2-dependent pathway

Angiotensin II (Ang II) plays a profound regulatory effect on NADPH oxidase and the functional features of vascular adventitial fibroblasts, but its role in antioxidant enzyme defense remains unclear. This study investigated the effect of Ang II on expressions and activities of superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) in adventitial fibroblasts and the possible mechanism involved. Ang II decreased the expression and activity of CAT in a dose- and time-dependent manner, but not that of SOD and GPx. The effects were abolished by the angiotensin II type 1 receptor (AT1R) blocker losartan and AT1R small-interfering RNA (siRNA). Incubation with polyethylene glycol-CAT prevented the Ang II-induced effects on reactive oxygen species (ROS) generation and myofibroblast differentiation. Moreover, Ang II rapidly induced phosphorylation of ERK1/2, which was reversed by losartan and AT1R siRNA. Pharmacological blockade of ERK1/2 improved Ang II-induced decrease in CAT protein expression. These in vitro results indicate that Ang II induces ERK1/2 activation, contributing to the downregulation of CAT as well as promoting oxidative stress and adventitial fibroblast phenotypic differentiation in an AT1R-mediated manner.

Antioxidants modulate the antiproliferative effects of nitric oxide on vascular smooth muscle cells and adventitial fibroblasts by regulating oxidative stress

Background S-nitrosothiols (SNO) release nitric oxide (NO) through interaction with ascorbic acid (AA). However, little is known about their combined effect in the vasculature. The aim of this study was to investigate the effect of AA on SNO-mediated NO release, proliferation, cell cycle progression, cell death, and oxidative stress in vascular cells. Methods Vascular smooth muscle cells and adventitial fibroblasts harvested from the aortae of Sprague–Dawley rats were treated with AA, ± S-nitrosoglutathione (GSNO), or ± diethylenetriamine NONOate (DETA/NO). NO release, proliferation, cell cycle progression, cell death, and oxidative stress were determined by the Griess reaction, [ 3H]-thymidine incorporation, flow cytometry, trypan blue exclusion, and 5-(and-6)chloromethyl-2′,7′dichlorodihydrofluorescein staining, respectively. Results AA increased NO release from GSNO 3-fold ( P < .001). GSNO and DETA/NO significantly decreased proliferation, but AA abrogated this effect ( P < .05). Mirroring the proliferation data, changes in cell cycle progression induced by GSNO and DETA/NO were reversed by the addition of AA. GSNO- and DETA/NO-mediated increases in oxidative stress were significantly decreased by the addition of AA ( P < .001). Conclusions Despite causing increased NO release from GSNO, AA reduced the antiproliferative and cell cycle effects of GSNO and DETA/NO through the modulation of oxidative stress.

ETA and ETB receptors are expressed in vascular adventitial fibroblasts

The adventitia has been recognized to play important roles in vascular oxidative stress, remodeling, and contraction. We recently demonstrated that adventitial fibroblasts are able to express endothelin (ET)-1 in response to ANG II. However, it is unclear whether ET-1 receptors are expressed in the adventitia. We therefore investigated the expression and roles of both ET(A) and ET(B) receptors in collagen synthesis and ET-1 clearance in adventitial fibroblasts. Adventitial fibroblasts were isolated and cultured from the mouse thoracic aorta by the explant method. Cultured cells were treated with ANG II (100 nmol/l) or ET-1 (10 pM) in the presence or absence of the ANG II type 1 receptor antagonist losartan (100 μM), the ET-1 receptor antagonists BQ-123 (ET(A) receptor, 1 μM) and BQ-788 (ET(B) receptor, 1 μM), and the ET(B) receptor agonist sarafotoxin 6C (100 nM). ET-1 peptide levels were determined by ELISA, whereas ET(A), ET(B), and collagen levels were determined by Western blot analysis. ANG II increased ET-1 peptide levels in a time-dependent manner. ANG II increased ET(A) and ET(B) receptor protein levels as well as collagen in a similar fashion. ANG II-induced collagen was reduced while in the presence of BQ-123, suggesting a role for the ET(A) receptor in the regulation of the extracellular matrix. ANG II treatment in the presence of BQ-788 significantly increased ET-1 peptide levels. Conversely, the ET(B) receptor agonist sarafotoxin 6C significantly decreased ET-1 peptide levels. These data implicate a role for the ET(B) receptor in the clearance of the ET-1 peptide. In conclusion, both ET(A) and ET(B) receptors are expressed in adventitial fibroblasts, which paves the ground for the biological significance of adventitial ET-1. The ET(A) receptor subtype mediates collagen I expression, whereas the ET(B) receptor subtype may play a protective role through increasing the clearance of the ET-1 peptide.

Expression and Function of Osteopontin in Vascular Adventitial Fibroblasts and Pathological Vascular Remodeling

Osteopontin is known to play important roles in various diseases including vascular disorders. However, little is known about its expression and function in vascular adventitial fibroblasts. Adventitial fibroblasts have been shown to play a key role in pathological vascular remodeling associating with various vascular disorders. In this study, we measured activation of Osteopontin and its biological functions in cultured adventitial fibroblasts and injured rat carotid injury arteries induced by balloon angioplasty. Our results showed that angiotensin II and aldosterone increased Osteopontin expression in adventitial fibroblasts in a time- and concentration-dependent manner. MAPKs and AP-1 pathways were involved in Osteopontin upregulation. In addition, Adventitial fibroblast migration stimulated by Angiotensin II and aldosterone required OPN expression. Perivascular delivery of antisense oligonucleotide for Osteopontin suppressed neointimal formation post-injury. We concluded that upregulation of Osteopontin expression in adventitial fibroblasts might be important in the pathogenesis of vascular remodeling after arterial injury.

Podoplanin-Positive Fibroblasts Enhance Lung Adenocarcinoma Tumor Formation: Podoplanin in Fibroblast Functions for Tumor Progression

During the metastatic process, cancer cells interact with vascular adventitial fibroblasts (VAF), which are the main components of the outermost connective tissue layer of blood vessels. This activity suggests the presence of a specific tumor microenvironment in the perivascular area. The s.c. coinjection of human lung adenocarcinoma cell lines (A549, PC-14, and CRL-5807) and human VAF (hVAF) resulted in a high rate of tumor formation, compared with the coinjection of these cell lines and human lung tissue-derived fibroblasts (hLF). A cDNA microarray analysis revealed a higher expression level of podoplanin in hVAFs than in hLFs (4.7-fold). Flow cytometry analysis also showed a higher expression level of podoplanin in hVAFs (43% ± 17.5%) than in hLFs (16% ± 10.3%). Sorted podoplanin-positive hVAFs displayed enhanced tumor formation, lymph node metastasis, and lung metastasis of A549 compared to sorted podoplanin-negative hVAFs. Knockdown of podoplanin in hVAFs decreased the augmenting effect of tumor formation and in vitro colony formation. The overexpression of podoplanin in hVAFs hastened the tumor formation of A549, compared with control hVAFs. Furthermore, the analysis of small-sized human lung adenocarcinoma (n = 112) revealed that patients with podoplanin-positive cancer-associated fibroblasts had a significantly higher rate of lymph node metastasis and a high risk of recurrence. These results indicate a promotive effect of hVAFs mediated by podoplanin on cancer progression and suggest that the perivascular environment may constitute a specific niche for tumor progression.

Angiotensin II downregulates catalase expression and activity in vascular adventitial fibroblasts through an AT1R/ERK1/2-dependent pathway

Angiotensin II (Ang II) plays a profound regulatory effect on NADPH oxidase and the functional features of vascular adventitial fibroblasts, but its role in antioxidant enzyme defense remains unclear. This study investigated the effect of Ang II on expressions and activities of superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) in adventitial fibroblasts and the possible mechanism involved. Ang II decreased the expression and activity of CAT in a dose- and time-dependent manner, but not that of SOD and GPx. The effects were abolished by the angiotensin II type 1 receptor (AT1R) blocker losartan and AT1R small-interfering RNA (siRNA). Incubation with polyethylene glycol-CAT prevented the Ang II-induced effects on reactive oxygen species (ROS) generation and myofibroblast differentiation. Moreover, Ang II rapidly induced phosphorylation of ERK1/2, which was reversed by losartan and AT1R siRNA. Pharmacological blockade of ERK1/2 improved Ang II-induced decrease in CAT protein expression. These in vitro results indicate that Ang II induces ERK1/2 activation, contributing to the downregulation of CAT as well as promoting oxidative stress and adventitial fibroblast phenotypic differentiation in an AT1R-mediated manner.

Nitric Oxide Increases Lysine-48-Linked Ubiquitination Of Cellular Proteins

Introduction: Proteins are targeted for degradation by the 26S proteasome by the addition of a polyubiquitin chain. Ubiquitin molecules can be linked through different lysine residues. The lysine 48 linkage (K48-Ub) is associated with protein degradation while the lysine 63 linkage (K63-Ub) is associated with intracellular signaling. Nitric oxide (NO) has many vasoprotective properties, including inhibition of neointimal hyperplasia, and we have previously shown that NO inhibits the ubiquitin-conjugating enzymes that regulate cell cycle progression. The aim of this current study is to characterize the effect of arterial injury on K48 or K63 ubiquitin bonds, with and without exposure to NO. Our hypothesis is that injury will increase, while NO will decrease, K48-linked ubiquitination of cellular proteins. Methods: The carotid injury model was performed in 11-wk male Sprague Dawley rats. Treatment groups included control, injury, injury+NO (PROLI/NO, 10mg), and NO alone (n=3/group). Arteries were harvested at 2 wks for immunohistochemical analysis and blinded grading of K48-Ub and K63-Ub expression (scale 0-3). Arteries were also harvested after 2, 24 and 72 hrs and homogenized for protein analysis. Vascular smooth muscle cells (VSMC) and adventitial fibroblasts (AF) were exposed to the NO donor DETA/NO (0.125-1 mM). Protein expression was determined by Western blot analysis, or immunoprecipitation (IP) and Western blot analysis. Results: Minimal expression of K48-Ub and K63-Ub was observed in uninjured control arteries (0±0). Injury increased K48-Ub in the neointima (3.0±0.0) and adventitia (2.0±0.2). NO decreased K48-Ub in the neointima (1.0±0.2) following injury, but had no effect on the adventitia (2.2±0.1). Interestingly, NO alone increased K48-Ub in the adventitia (0.7±0.0). Changes in K63-Ub expression were smaller. Injury slightly increased K63-Ub in the neointima (1.1±0.4) and adventitia (0.7±0.2). NO slightly decreased K63-Ub in the neointima (0.3±0.2) following injury, but slightly increased K63-Ub in the adventitia (1.3±0.2). No changes were seen in the media in any treatment group. Analysis of carotid lystates showed that injury increased K48-Ub expression at all time points, while injury+NO further increased K48-Ub. Minimal changes in K63-Ub were seen among all groups. Analysis of VSMC showed an increase in K48-Ub and no change in K63-Ub expression with DETA/NO in vitro. In VSMC and AF, IP revealed that DETA/NO increased expression of K48-Ub-linked cyclin A and B but did not change expression of K63-Ub-linked cyclin A and B. Conclusions: Supporting our hypothesis, we have shown that injury increases K48-linked ubiquitination in vivo. Surprisingly, NO further increases K48-linked ubiquitination in vitro and in vivo. These changes are a remarkable contrast to K63-linked ubiquitination, where minimal changes were seen. These novel findings provide important new insights into the mechanism by which NO prevents neointimal hyperplasia by regulating the ubiquitin-proteasome pathway.

E0156 Urotensin II promotes monocyte chemoattractant protein-1 expression in aortic adventitial fibroblasts of rat

BackgroundRecent studies reported that vascular adventitial fibroblasts (AFs) are involved in the development of vascular inflammatory diseases, such as atherosclerosis. Urotensin II (UII), a potent vasoconstrictive peptide, could stimulate phenotype...

E0545 Research on inhibiting vascular remodelling of Captopril and Xuefuzhuyu Capsule on the basis of restraining activation and migration of adventitial fibriblasts

BackgroundVascular remodelling is the common pathophysiological basis of artherosclerosis, hypertension and coronary artery disease, et al. It is also one of pathogenesis mechanisms which causes target organs damage and dysfunction....

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

To identify proteins that could potentially be involved in adventitial remodeling in vascular adventitial fibroblasts (AFs) from spontaneously hypertensive rats (SHR). 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. 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. 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.