RGDechiHCit was prepared for the in vitro and in vivo studies as previously described . To test the biological effects of RGDechiHCit, we synthesized the cyclic pentapeptide c(RGDf[NMe]V), also known as cilengitide or EMD 121974 [14, 19]. We also investigated RGDechiHCit and c(RGDf[NMe]V) peptides degradation in serum. Both peptides were incubated and the resulting solutions were analyzed by liquid chromatography/mass spectrometry (LC/MS) at different times. 20μL of human serum (Lonza, Basel, Switzerland) were added to 8 μL of a 1 mg/ml solution of either RGDechiHCit or c(RGDf[NMe]V) at 37°C. After 1, 2, 4 and 24h, samples were centrifuged for 1min at 10000g. Solutions were analyzed by LCQ Deca XP Max LC/MS system equipped with a diode-array detector combined with an elctrospray ion source and ion trap mass analyzer (ThermoFinnigan, San Jose, CA, USA), using a Phenomenex C18 column (250× 2 mm; 5μm; 300 Ǻ) and a linear gradient of H2O (0.1%TFA)/CH3CN (0.1%TFA) from 10 to 80% of CH3CN (0.1%TFA) in 30 min at flow rate of 200μL/min.
In vitro studies
In vitro studies were performed on cell cultures of ECs or VSMCs, cultured in Dulbecco's modified Eagle's medium (DMEM; Sigma-Aldrich, Milan, Italy) as previously described and validated [22, 23]. Cell culture plates were filled with 10 μg/cm2 of human fibronectin (hFN, Millipore®, Bedford, MA, USA) as described . All experiments were performed in triplicate with cells between passages 5 and 9.
Cell proliferation assay
Cell cultures were prepared as previously described . Briefly, cells were seeded at density of 100000 per well in six-well plates, serum starved, pre-incubated at 37°C for 30' with c(RGDf[NMe]V) or RGDechiHCit (10-6 M). Proliferation was induced using hFN (100 μg/ml). Cell number was measured at 3, 6 and 20 h after stimulation as previously described [26, 27].
DNA synthesis was assessed as previously described . Briefly, cells were serum-starved for 24 h and then incubated in DMEM with [3H]thymidine and 5% FBS. After 3, 6 and 20 h, cells were fixed with trichloracetic acid (0.05%) and dissolved in 1M NaOH. Scintillation liquid was added and [3H]thymidine incorporation was assessed as previously described .
VEGF production was measured as previously described . Briefly, ECs were seeded at a density of 600000 per well in six well plates, serum starved overnight, seeded with c(RGDf[NMe]V) or RGDechiHCit (10-6 M) and then stimulated with hFN for 6 hours. Cultured medium was collected and VEGF production was revealed by western blot.
Endothelial Matrigel assay
The formation of network-like structures by ECs on an extracellular matrix (ECM)-like 3D gel consisting of Matrigel® (BD Biosciences, Bedford, MA, USA), was performed as previously described and validated [27, 28]. The six-well multidishes were coated with growth factor-reduced Matrigel in according to the manufacturer's instructions. ECs (5×104) were seeded with c(RGDf[NMe]V) or RGDechiHCit (10-6 M), in the absence (negative control) or presence (100 μg/ml) of hFN . Cells were incubated at 37°C for 24h in 1 ml of DMEM. After incubation, ECs underwent differentiation into capillary-like tube structures. Tubule formation was defined as a structure exhibiting a length four times its width . Network formation was observed using an inverted phase-contrast microscope (Zeiss). Representative fields were taken, and the average of the total number of complete tubes formed by cells was counted in 15 random fields by two independent investigators.
Immunoblot analyses were performed as previously described and validated [23, 28]. Mouse monoclonal antibodies to extracellular signal regulated kinase (ERK2) and phospho-ERK, anti-rabbit VEGF and actin were from Santa Cruz Biotecnology (Santa Cruz, CA, USA). Levels of VEGF were determined using an antibody raised against VEGF-165 (Santa Cruz Biotechnology) . Experiments were performed in triplicate to ensure reproducibility. Data are presented as arbitrary densitometry units (ADU) after normalization for the total corresponding protein or actin as internal control .
In vivo studies
Wound healing assay was performed on 14-week-old (weight 293 ± 21 g) normotensive WKY male rats (Charles River Laboratories, Calco (LC), Italy; n = 18), and Matrigel plugs experiments were carried out on 16-week-old (weight 33 ± 4 g) c57BL/6 mice (Charles River Laboratories, Milan, Italy; n = 13). All animal procedures were performed in accordance with the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health in the United States (NIH Publication No. 85- 23, revised 1996) and approved by the Ethics Committee for the Use of Animals in Research of "Federico II" University .
The rats (n = 18) were anesthetized using vaporized isoflurane (4%, Abbott) and maintained by mask ventilation (isoflurane 1.8%). The dorsum was shaved by applying a depilatory creme (Veet, Reckitt-Benckiser, Milano, Italy) and disinfected with povidone iodine scrub. A 20 mm diameter open wound was excised through the entire thickness of the skin, including the panniculus carnosus layer, as described and validated [1, 28]. Pluronic gel (30%) containing (10-6 M) c(RGDf[NMe]V) (n = 6), RGDechiHCit (n = 7), or saline (n = 5) was placed daily directly onto open wounds, then covered with a sterile dressing. Two operators blinded to the identity of the sample examined and measured wound areas every day, for 8 days. Direct measurements of wound region were determined by digital planimetry (pixel area), and subsequent analysis was performed using a computer-assisted image analyzer (ImageJ software, version 1.41, National Institutes of Health, Bethesda, MD, USA). Wound healing was quantified as a percentage of the original injury size. Eight days after wounding, rats were euthanized. Wounds did not show sign of infection. The lesion and adiacent normal skin were excised, fixed by immersion in phosphate buffered saline (PBS, 0.01 M, pH 7.2-7.4)/formalin and then embedded in paraffin to be processed for immunohistology, as described .
Mice (n = 13), anesthetized as described above, were subcutaneously injected midway on the dorsal side, using sterile conditions, with 0.2 ml of Matrigel® basement matrix, pre-mixed with 10-6M VEGF and 10-5M c(RGDf[NMe]V) (n = 4), 10-6M VEGF and 10-5M RGDechiHCit (n = 5), or 10-6M VEGF alone (n = 4). After seven days, mice were euthanized and the implanted plugs were harvested from underneath the skin, fixed in 10% neutral-buffered formalin solution and then embedded in paraffin. Invading ECs were identified and quantified by analysis of lectin immunostained sections, as described [1, 2].
All tissues were cut in 5 μm sections and slides were counterstained with a standard mixture of hematoxylin and eosin. For Masson's trichrome staining of collagen fibers, useful to assess the scar tissue formation, slides were stained with Weigert Hematoxylin (Sigma-Aldrich, St. Louis, MO, USA) for 10 minutes, rinsed in PBS (Invitrogen) and then stained with Biebrich scarlet-acid fuchsin (Sigma-Aldrich) for 5 minutes. Slides were rinsed in PBS and stained with phosphomolybdic/phosphotungstic acid solution (Sigma-Aldrich) for 5 minutes then stained with light green (Sigma-Aldrich) for 5 minutes . ECs were identified by lectin immunohistochemical staining (Sigma-Aldrich)  and quantitative analysis was performed using digitized representative high resolution photographic images, with a dedicated software (Image Pro Plus; Media Cybernetics, Bethesda, MD, USA) as previously described .
Data presentation and statistical analysis
All data are presented as the mean value ± SEM. Statistical differences were determined by one-way or two-way ANOVA and Bonferroni post hoc testing was performed where applicable. A p value less than 0.05 was considered to be significant. All the statistical analysis and the evaluation of data were performed using GraphPad Prism version 5.01 (GraphPad Software, San Diego, CA, USA).