Serum samples were obtained from 48 patients with SSc (43 female and 5 male patients; median age 64 years, range 37 to 78 years), defined as limited cutaneous SSc (lcSSc; n = 36) or diffuse cutaneous SSc (dcSSc; n = 12) , 45 subjects with pRP and 48 age-matched and sex-matched healthy controls. All SSc patients reported the occurrence of RP. Nailfold videocapillaroscopy (NVC) was performed on 10 fingers and all SSc patients were classified as having early, active and late NVC patterns . At the time of blood collection, the presence of DUs was recorded. DUs related to trauma or calcinosis were not included in the analyses. The patients enrolled were not on corticosteroids, immunosuppressants or other disease-modifying drugs or calcium channel blockers. Peripheral blood samples were collected without any additive, left to clot for 30 minutes before centrifugation at 1,500 g for 15 minutes, and serum was collected and stored in aliquots at −80 °C until used. Paraffin-embedded sections of lesional forearm skin biopsies were obtained from 14 patients with SSc (12 women and 2 men, n = 9 with lcSSc and n = 5 with dcSSc; median age 46.8 years, range 27 to 69 years, and median disease duration 6 years, range 1 to 16 years) and 12 age-matched and sex-matched healthy donors. The study was approved by the Ethical Committee of the Azienda Ospedaliero-Universitaria Careggi (AOUC), Florence, Italy, and all subjects provided written informed consent.
Isolation of dermal microvascular endothelial cells (MVECs) and cell culture
Human dermal MVECs were isolated from skin biopsies of five SSc patients (SSc-MVECs) and five healthy subjects (H-MVECs). Briefly, the samples were mechanically cleaned to remove the adipose and epidermis layers, in order to obtain a pure specimen of vascularized dermis, and were treated as previously described . The samples were placed at 37 °C in a humidified atmosphere with 5 % CO2. After one day of culture in endothelial cell basal medium (EBM-2, catalog number LOCC3156; Euroclone, Milan, Italy) supplemented with 20 % fetal bovine serum (FBS), 5 ng/ml H-epidermal growth factor (hEGF; Clonetics Corporation, San Diego, California, USA), 1 μg/ml hydrocortisone acetate, 100 U/ml penicillin, 100 μg/ml streptomycin, and 25 μg/ml amphotericin B without addition of further angiogenic growth factors, small colonies of polygonal elements were detected. Non-adherent cells were removed and fresh endothelial cell complete medium was added. To maintain optimal culture conditions, media were changed every third day, and after 2 weeks of primary culture a monolayer of cells was obtained. MVECs from primary cultures were further identified using immunomagnetic beads recognizing CD31. Isolated cells were purified as MVECs by labeling with anti-factor VIII-related antigen and anti-CD105, followed by reprobing with anti-CD31 antibodies. Dermal MVECs were maintained in endothelial cell complete medium and were used between the third and seventh passages in culture.
ELISA on serum samples
Serum levels of Sema3E protein were measured by a colorimetric sandwich enzyme-linked immunosorbent assay (ELISA kit, catalogue number ABIN1117868; Antibodies Online, Aachen, Germany), according to the manufacturer’s instructions. Briefly, standards and samples (100 μl/well) were added to the appropriate 96-well microtiter plate pre-coated with a biotin-conjugated polyclonal antibody specific for the short basic Sema3E domain, and were incubated for 2 hours at 37 °C. Subsequently the samples and standard were removed and 100 μl of Detection Reagent A working solution were added to each well for 1 hour at 37 °C. The microplates were washed three times with wash solution, followed by the addition of 100 μl of Detection Reagent B working solution (Avidin-conjugated Horseradish Peroxidase (HRP)) to each well, and incubation for 30 minutes at 37 °C. The microplates were washed five times and the reaction was developed in the dark with 90 μl of substrate solution (tetramethylbenzidine) and then stopped by applying 50 μl of sulfuric acid (1 M H2SO4). The absorbance of each well was read using a microplate reader at 450 nm. Serum levels of Sema3E were read from a standard curve prepared using a lyophilized protein standard reconstituted with standard diluent included in the kit. The detection range of the assay was 0.156−20 ng/ml. Each sample was measured in duplicate. Serum Sema3E concentration was determined by comparing the optical density (OD) of each sample to the standard curve.
Immunohistochemistry and fluorescence microscopy
Paraffin-embedded skin sections (5 μm thick) were deparaffinized and boiled for 10 minutes in sodium citrate buffer (10 mM, pH 6.0) in order to expose antigens. After three washes in phosphate-buffered saline (PBS), the sections were incubated in 2 mg/ml glycine for 10 minutes to quench autofluorescence due to free aldehydes, and then blocked for 1 hour at room temperature with 1 % bovine serum albumin (BSA) in PBS. The samples were then incubated overnight at 4 °C with goat anti human Sema3E antibody (catalog number ab112886; Abcam, Cambridge, UK) diluted 1:100 in PBS with 1 % BSA. After extensive washing in PBS, the sections were incubated with Alexa Fluor-488-conjugated donkey anti-goat IgG for 45 minutes at room temperature in the dark (1:200 dilution; Invitrogen, San Diego, CA, USA). For double immunofluorescence staining, we used a rabbit polyclonal antibody against CD31/platelet-endothelial cell adhesion molecule-1 (PECAM-1) diluted 1:50 (catalog number ab28364; Abcam), followed by Alexa Fluor-568-conjugated donkey anti-rabbit IgG (1:200 dilution; Invitrogen). To evaluate PlxnD1 expression, skin sections were incubated overnight at 4 °C with rabbit polyclonal antibody against human PlxnD1 (catalog number ab28762; Abcam) diluted 1:50 in PBS with 1 % BSA. After extensive washing in PBS, the section were processed as described above, and subsequently incubated with Alexa Fluor-488-conjugated goat anti-rabbit IgG (1:200 dilution; Invitrogen) for 45 minutes at room temperature in the dark. For double immunofluorescence staining with anti-PlxnD1 antibody, we used a mouse monoclonal anti-CD31 antibody (1:20 dilution; catalog number M0823; Dako, Glostrup, Denmark) followed by Rhodamine Red-X-conjugated goat anti-mouse IgG (1:200 dilution; Invitrogen). Irrelevant isotype-matched and concentration-matched goat, mouse, and rabbit IgG (Sigma-Aldrich, St Louis, MO, USA) were used as negative controls. Nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) (Chemicon International, Temecula, CA, USA). The immunolabeled sections were then observed under a Leica DM4000 B microscope equipped with fully automated fluorescence axes (Leica Microsystems, Mannheim, Germany). Fluorescence images were captured using a Leica DFC310 FX 1.4-megapixel digital colour camera equipped with the Leica software application suite LAS V3.8 (Leica Microsystems). Densitometric analysis of the intensity of immunofluorescent staining was performed on digitized images using the free-share ImageJ software (NIH, Bethesda, MD, USA; online at ).
Western blotting and cell signaling
Total proteins were extracted from dermal H-MVECs and SSc-MVECs according to standard protocols. In some experimental conditions, before protein extraction H-MVECs were cultured for 24 hours in EBM-2 medium containing 10 % of serum from healthy subjects (n = 5) and SSc patients (n = 5) or recombinant human VEGF-A165 (10 ng/ml; R&D Systems, Minneapolis, MN, USA). As determined by ELISA, healthy and SSc serum samples containing Sema3E levels similar to the respective median group value were used in these experiments. Twenty-five micrograms of total proteins were electrophoresed on NuPAGE 4 to 12 % Bis-Tris Gel (Invitrogen) and were blotted on polyvinylidene difluoride membranes (Invitrogen). The membranes were blocked for 40 minutes at room temperature with the blocking solution included in the Western Breeze Chromogenic Western Blot Immunodetection Kit (Invitrogen) on a rotary shaker and incubated for 1 hour at room temperature with rabbit polyclonal antihuman PlxnD1 (Sema domain 250 kDa, catalog number PP4401; 1:1000 dilution; ECM Biosciences, Versailles, Woodford County, KY, USA), rabbit polyclonal antihuman PlxnD1 (Cytoplasmic domain 250 kDa, catalog number PP4421; 1:500 dilution; ECM Biosciences), rabbit polyclonal antihuman PlxnD1 (a.a. 1635–1647 C-ter region 250 kDa, catalog number PP4441; 1:500 dilution; ECM Biosciences), rabbit polyclonal antihuman Sema3E (N-terminal region 87 kDa, catalog number SP4461; 1:1000 dilution; ECM Biosciences) and rabbit polyclonal anti-α-tubulin (catalog number ab18251; 1:1000 dilution; Abcam) antibodies, assuming α-tubulin as control invariant protein. Immunodetection was performed as described in the Western Breeze Chromogenic Immunodetection protocol (Invitrogen). ImageJ software (NIH) was used for densitometric analysis of the bands and all values were normalized to α-tubulin.
In vitro capillary morphogenesis assay
In vitro capillary morphogenesis assay was performed in 96-well plates covered with Matrigel (BD Biosciences, Milan, Italy). Matrigel (50 μl; 10–12 mg/ml) was pipetted into culture wells and polymerized for 30 minutes to 1 hour at 37 °C, as described elsewhere . H-MVECs and SSc-MVECs (30 × 103 cells/well) were incubated in EBM-2 medium containing 10 % FBS. In some experimental conditions, H-MVECs were incubated in EBM-2 medium with 10 % serum from healthy subjects (n = 5), SSc patients (n = 5) and pRP subjects (n = 5), alone or in combination with human Sema3E peptide (100 ng/ml; catalog number ab39320; Abcam), human PlxnD1 peptide (a.a. 1683–1694) (100 ng/ml; catalog number ab45701; Abcam) or both. As determined by ELISA, healthy, pRP and SSc serum samples containing Sema3E levels similar to the respective median group value were used in these experiments. Stimulation with recombinant human VEGF-A165 (10 ng/ml; R&D Systems) was used as positive control of angiogenesis. Plates were photographed at 6 and 24 hours. Results were quantified at 24 hours by measuring the percent field occupancy of capillary projections, as determined by image analysis. Six to nine photographic fields from three plates were scanned for each experimental point.
Data are expressed as the mean ± standard deviation (SD) or median and range. The Student’s t test and nonparametric Mann–Whitney U test were used where appropriate for statistical evaluation of the differences between two independent groups. A p value less than 0.05 was considered statistically significant.