C57BL/6 mice were purchased from The Jackson Laboratory. All of the experiments were performed under the guidelines of the Boston University Institutional Animal Care and Use Committee (protocol AN-15037). Alzet osmotic miniature pumps (model 2002) delivering angiotensin II (Sigma-Aldrich, St. Louis, MO, USA) at a rate of 1,000 ng/kg/min (pressor dose) or 2,000 ng/kg/min, or PBS, were implanted subcutaneously on the backs of 8-week-old mice. After 14 days, mice were killed, and the skin surrounding the pump outlet was collected by using an 8-mm-diameter punch biopsy device.

Gomori Trichrome staining was used to detect collagen fibers and collagen deposition in the mouse skin. The skin samples were fixed in 4% paraformaldehyde for 24 hours and then processed for paraffin embedding. Staining was performed on 8-μm-thick paraffin sections by following the manufacturer's instructions (Chromaview, Dublin, OH, USA; Gomori Trichrome blue collagen Kit S7440-19). Collagen fibers were stained blue, nuclei were stained black, and the background was stained red.

Collagen deposition was quantified by measuring total hydroxyproline content in 4-mm skin-punch biopsies obtained from PBS and Ang II infusion sites by using a previously described method with some modifications 17. In brief, the skin samples were hydrolyzed with 6 M sodium hydroxide at 110°C for 12 hours. The hydrolyzate was then oxidized with oxidation buffer (one part 7% chloramine T and four parts of acetate citrate buffer) for 4 minutes at room temperature. Ehrlich aldehyde reagent was added to each sample, and the chromophore was developed by incubating the samples at 65°C for 25 minutes. Absorbance of each sample was read at 560 nm by using a spectrophometer. Results were expressed as total hydroxyproline content (in micrograms) per 0.1 g of tissue. A standard curve was performed for all hydroxyproline measurements by using known quantities of hydroxyproline.

Total RNA was isolated by using the RNeasy Fibrous Tissue Mini Kit (Qiagen, Valencia, CA, USA). Then, 1 μg of total RNA was reverse transcribed with random hexamers by using the Transcriptor First Strand Complementary DNA Synthesis kit (Roche Applied Science, Indianapolis, IN, USA) according to the manufacturer's protocol. Real-time PCR assays were performed by using the StepOnePlus Real-Time PCR system (Applied Biosystems, Foster City, CA, USA). The amplification mixture (10 μl) contained 1 μl of complementary DNA, 0.5 μM of each primer, and 5 μl of SYBR Green PCR Master Mix. The primers are listed in Supplementary Table 1. Relative change in the levels of genes of interest was determined by the 2^-ΔΔCT method.

For all immunofluorescence staining, skin samples were directly embedded in O.C.T. compound, flash frozen, and stored at -80°C. Staining was performed on 8-μm cryosections of mouse skin. In brief, slides were blocked with a blocking solution (3% BSA (Sigma-Aldrich), and 0.3% Triton X-100 in PBS) for 2 hours. After washing, tissue sections were incubated at 4°C overnight with primary antibodies (Table 2). Tissue sections were then washed and incubated with secondary Ab (Table 2) at room temperature for 2 hours. Coverslips were mounted by using Vectashield with DAPI (Vector Laboratories, Burlingame, CA, USA), and staining was examined by using a FluoView FV10i confocal microscope system (Olympus, Center Valley, PA, USA) at 488 nm (green), 594 nm (red), and 405 nm (blue).

Human dermal microvascular endothelial cells (HDMECs) were isolated from human foreskin, as previously described 18. Cells were cultured on bovine collagen-coated six-well plates in EBM medium supplemented with 10% FBS and EC growth supplement mix at 37°C with 5% CO₂ in air. The culture medium was changed every other day. For immunofluorescence, cultured HDMECs grown on collagen-coated coverslips were treated with Ang II (1,000 ng/ml) for 96 hours. Control and Ang II-treated cells were fixed with 4% paraformaldehyde for 15 minutes followed by incubation with 0.15 M glycine for 30 minutes. Nonspecific protein binding was blocked with 3% BSA for 1 hour. Next, cells were incubated at 4°C overnight with primary antibodies: goat anti-mouse VE-cadherin (Santa Cruz Biotechnology, Santa Cruz, CA, USA), and rabbit anti-mouse FSP1 (Abcam, Cambridge, MA, USA). After washing, cell cultures were incubated with Alexa fluor 488 donkey anti-goat (Invitrogen, Grand Island, NY, USA) and Alexa fluor 594 donkey anti-rabbit (Invitrogen) antibodies for 1.5 hour. Cells were mounted on slides by using Vectashield with DAPI (Vector Laboratories) and examined by using a FluoView FV10i confocal microscope system (Olympus, Center Valley, PA, USA) at 488 nm (green), 594 nm (red), and 405 nm (blue).

Immunohistochemistry was performed on formalin-fixed, paraffin-embedded skin tissue sections by using the Vectastain ABC kit (Vector Laboratories) according to the manufacturer's instructions. In brief, sections (8-μm thick) were mounted on APES (aminopropyltriethoxy silane solution)-coated slides, deparaffinized with Histo-Clear (National Diagnostics, Atlanta, GA, USA), and rehydrated through a graded series of ethanol. Endogenous peroxidase was blocked by incubation in 3% hydrogen peroxide for 30 minutes, followed by incubation with 0.15 M glycine for 45 minutes, and normal blocking serum for 1 hour. The sections were then incubated overnight at 4°C with antibodies against CD3 (Abcam, Cambridge, MA, USA), Mac3 (BD Bioscience, San Jose, CA, USA), CD45R (AbD Serotec, Raleigh, NC, USA), or CD163B (Epitomics, Burlingame, CA, USA), diluted 1:100 in blocking buffer, followed by incubation for 30 minutes with a biotinylated secondary antibody solution. A solution containing avidin:biotin:peroxidase complexes was applied to the sections subsequently. Immunoreactivity was visualized with diaminobenzidine (Vector Laboratories), and the sections were counterstained with hematoxylin. Images were collected by using a microscope (BH-2; Olympus, Center Valley, PA, USA).

All data were analyzed with the Student paired t test. The level for statistical significance was set at P ≤ 0.05.

To determine whether angiotensin II can induce dermal fibrosis, Ang II at a rate of 1,000 ng/kg/min was administered continuously with subcutaneous osmotic minipumps implanted under the shaved back skin of 8-week-old C57BL/6 male mice. Treated skin was harvested at day 14, and histologic examination and collagen-content measurement assays were performed. Gomori Trichrome staining showed that collagen fibers were more closely packed in angiotensin II-treated mice compared with control mice, reflecting increased collagen deposition (Figure 1A). In addition, the fat layer of the reticular dermis was partially replaced with extracellular matrix (Figure 1A). Real-time PCR analysis showed a statistically significant increase in mRNA levels of the collagen-encoding genes: Col1a1, Col1a2, Col3a1, and Col5a1, as well as CTGF (2.4-fold, 1.9-fold, and 3.4-fold, 1.9-fold, and 2.1-fold, respectively; *P ≤ 0.05) in the skin of Ang II-infused mice when compared with control mice (Figure 1B). Total hydroxyproline content from the injected sites was significantly higher in Ang II (1,000 ng/kg/min)-infused skin (2,481 ± 920 μg per 0.1 g of skin) compared with control PBS-infused skin (1,534 ± 430 μg per 0.1 g of skin; *P ≤ 0.05) (Figure 1C). A higher dose of Ang II (2,000 ng/kg/min) showed a larger increase in the total hydroxyproline content of local skin (5,272 ± 1,260 μg per 0.1 g of Ang II-infused skin versus 1,748 ± 531 μg per 0.1 g of PBS-infused skin; *P ≤ 0.05) (Figure 1C). The profibrotic effects of Ang II were local, as no significant differences were observed in hydroxyproline content in distal skin with either concentration of Ang II (data not shown). Because a dose of 1,000 ng/kg/min was sufficient to induce significant dermal fibrosis, this dose was selected for further analyses.

Transforming growth factor-β (TGF-β) plays a pivotal role in the pathogenesis of scleroderma by activating fibroblasts and stimulating the production of ECM components 19. To determine whether Ang II treatment activates the TGF-β pathway, we examined skin samples from the infused sites with real-time PCR and immunostaining. Real-time PCR analysis showed statistically significant increases in the mRNA levels of TGF-β2 and TGF-β3 (3.2-fold and 4.8-fold, respectively; *P ≤ 0.05) in Ang II-treated mouse skin (Figure 2A). No differences were observed in the mRNA expression of TGF-β1 (Figure 2A). Immunohistochemical staining of pSmad2 was performed on paraffin sections. We observed increased numbers of pSmad2-positive cells distributed throughout all dermal layers in Ang II-treated mice (Figure 2B). These data suggest that Ang II potently activates TGF-β signaling in this model.

Fibrosis is associated with accumulation of activated fibroblasts/myofibroblasts in the affected tissues. To determine whether Ang II treatment increases myofibroblast presence, skin from infused sites was examined with real-time PCR and immunostaining for αSMA. Real-time PCR analysis showed a statistically significant increase in the mRNA level of αSMA (1.9-fold, *P ≤ 0.05) in Ang II-treated mouse skin (Figure 3A). αSMA staining was performed on cryosections (immunofluorescence, Figure 3B and 3D) and paraffin sections (immunohistochemistry, Figure 3C) from PBS- and Ang II-treated mouse skin. Affected sites from PBS-treated mice contained few αSMA-positive cells, observed exclusively around the blood vessels and the arrector pili muscles. In contrast, affected sites from Ang II-treated mice showed an increased number of cells expressing αSMA distributed mainly throughout the upper dermis (Figure 3B and 3C), whereas in the lower dermis, positive cells were observed only around the blood vessels (Figure 3D). These data suggest that Ang II-induced myofibroblast differentiation contributes to the development of fibrosis in this model.

Previous studies have shown that Ang II promotes inflammation in kidney and heart models of fibrosis 56. To evaluate the recruitment of inflammatory cells, skin samples from the Ang II-infused sites were examined with real-time PCR and immunostaining of immune cell markers. Real-time PCR analysis showed statistically significant increases in the mRNA levels of the T-cell marker, CD3, B-cell marker, CD45/B220, and the macrophage marker Emr1 (2.0-fold, 3.4-fold, and 3.6-fold, respectively; *P ≤ 0.05), in Ang II-treated mouse skin (Figure 4A and 5A). Immunohistochemical staining of CD3, CD45R, and two macrophage markers, a general marker, Mac3 20, and a marker identifying M2 macrophages, CD163B 2122 was performed on paraffin sections. We observed increased infiltration of CD3, CD45R, and Mac3-positive cells only in the hypodermis of Ang II-treated mice (Figure 4B and 5B). Interestingly, we observed an increased presence of CD163B-positive cells in the upper dermis of Ang II-treated mice, whereas no increase was found in the lower dermis or hypodermis (Figure 5C).

Circulating mesenchymal cells, fibrocytes, have been shown to infiltrate areas of inflammation and tissue damage and contribute to the fibrotic process 23. To determine whether fibrocytes are involved in the dermal fibrosis induced by Ang II, skin cryosections were stained for the immune cell marker, CD45, and mesenchymal markers, FSP1 and P4H. Skin samples from the Ang II-injected mice showed increased numbers of the CD45/FSP1 double-positive cells (3.83% ± 0.49% versus 0.94% ± 0.37% of total number of cells/HPF; *P ≤ 0.05) distributed mostly around small vessels in the lower dermis and around the sclerotic collagen bundles in the upper dermis (Figure 6A). Similar results were observed for the CD45/P4H double-positive cells (5.11% ± 0.6% versus 2.85% ± 0.75% of cells per HPF; *P ≤ 0.05; Figure 6A). In addition, although only a few FSP1-positive cells were seen in PBS-infused skin, a significant increase in FSP1 cells was observed in the upper dermis of Ang II-infused mice (Figure 6A). We also observed a 1.55-fold increase in FSP1 mRNA levels in circulating cells isolated from Ang II-treated mice; however, this difference was not statistically significant (data not shown). However, mRNA levels of FSP1 and of the inflammatory chemokine MCP1, a chemoattractant for fibrocytes, were significantly elevated (1.94-fold, 2.5-fold, respectively; *P < 0.05) in Ang II-treated mouse skin (Figure 6B). No differences were observed in the mRNA expression of another fibrocyte chemoattractant, SDF1/CXCL12 (data not shown).

Recent studies have indicated that EndoMT can contribute to the progression of fibrotic diseases, and Ang II has been implicated in this process 24252627. To determine whether EndoMT could serve as a source of activated fibroblasts, we performed double-fluorescence staining for VE-cadherin and FSP1 in the skin samples from Ang II- and PBS-treated mice (Figure 7A). Ang II-treated mice showed increased numbers of VE-cadherin/FSP1 double-positive cells distributed around small vessels in the lower dermis. No VE-cadherin/FSP1 double-positive cells were detected in the upper dermis. In a complementary experiment, cultured HDMECs treated with Ang II (Figure 7B) demonstrated decreased expression of the endothelial marker, VE-cadherin, and increased expression of the mesenchymal marker, FSP1.