Endothelial cell specific effect of 16K hPRL.

<p>The response of human brain vascular pericytes (HBVP) and human umbilical vein endothelial cells (HUVEC) to 16K hPRL was examined. (A) HBVP viability was evaluated by means of the calcein assay. 16K: 100 nM 16K hPRL. Cam: camptothecin-treated cells, a positive control. Rlu: relative luminescence units. Each bar represents a mean ± SEM, n = 3. Two different experiments were performed. (B) HBVP were treated with 20 ng/ml PDGF-BB to stimulate proliferation and 100 nM 16K hPRL (16K). HBVP proliferation was assayed 48 h later by measuring BrdU incorporation. The data presented are means ± SEM, n = 5 and are representative of at least two independent experiments. (C) HBVP migration was assessed in a modified Boyden chamber (Costar, Corning Inc.). HBVP were treated for 17 h with 20 ng/ml PDGF-BB to stimulate migration and 100 nM 16K hPRL (16K). The data presented are means ± SEM, n = 3 and are representative of at least two independent experiments. (D) Apoptosis in HUVEC was assessed by quantification of Caspase-3 activity. HUVEC were treated for 16h with 50 nM 16K hPRL. (E) HUVEC were treated with 10 ng/ml bFGF to stimulate proliferation and 100 nM 16K hPRL (16K). HUVEC proliferation was assayed 24 h later by measuring BrdU incorporation. The data presented are means ± SEM, n = 5 and are representative of at least two independent experiments. (F) HUVEC migration in a scratch-wound assay 8 h after treatment with bFGF and VEGFA and with or without 50 nM 16K hPRL. The data presented are means ± SEM, 5 measurements/condition, n = 3 and are representative of at least two independent experiments. n.s., non significant.*, P<0.05. **, P<0.001. ***, P<0.0001.</p

Decreased expression of Notch factors in HUVEC-HBVP cocultures after 16K hPRL treatment.

<p>In cultures of HBVP or HUVEC alone, the cells were treated with 100 nM 16K hPRL (16K) for 16 h. HBVP+HUVEC cocultures were seeded and treated as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0027318#pone-0027318-g006" target="_blank">Fig. 6</a>, but the time spent in coculture was longer (16 h). The two cell populations were then separated prior to qRT-PCR analysis. In HBVP, transcripts of <i>NOTCH3</i> and <i>aSMA</i> were detected (respectively A and C). Western blot analysis was also performed to detect Notch3 ICD (B). In HUVEC, <i>DLL1</i> transcripts were detected (D). In HBVP and HUVEC, <i>JAGGED1</i> transcripts were detected (respectively E and F). The presented data are means ± SEM, n = 3 and are representative of at least two independent experiments. n.s., non significant. ***, P<0.0001.</p

Decreased expression of Notch factors in HUVEC cocultured with HBVP after 16K hPRL treatment.

<p>(A–E) HUVEC pretreated for 30 min with 100 nM 16K hPRL (16K) were cultured for 6 h in the presence or absence of HBVP. The two cell populations were then separated prior to qRT-PCR analysis and subjected to qRT-PCR analysis to detect <i>DLL4</i>, <i>NOTCH4</i>, <i>NOTCH1</i>, <i>HEY2</i> and <i>EPHRINB2</i> transcripts. (F) Western blot analysis to detect Notch1 ICD. Data are presented as means ± SEM, n = 3 and are representative of at least two different experiments. n.s., non significant. *, P<0.05; ***, P<0.0001.</p

Inhibition of tumor growth by 16K hPRL is associated with an increased vessel number but with decreased vessel perfusion.

<p>(A) The data represent mean tumor volumes ± SEM in 16K-Ad-treated (n = 8) or Null-Ad-treated (n = 10) mice. (B) The number of tumor vessels per field ± SEM was determined on CD31-positive vessels in x100 power fields of each section. <i>Bar</i>, 100 µm. (C) Representative photographs of tumor sections from 16K-Ad- and Null-Ad-inoculated mice, double-stained for the endothelial cell marker CD31 (red) and the perfusion marker lectin (green). White arrows, vessels stained positive for CD31 and lectin. Heads of white arrows, vessels only positive for CD31. (D) The perfusion index was calculated as the percentage of lectin-positive vessels (perfused vessels) per CD31-positive vessels in the B16F10 tumors ± SEM. For (B) and (D) the data represent, for each set of conditions, the mean ratio ± SEM calculated for all the fields of all tumor sections (one section per tumor). *, P<0.05.</p

16K hPRL inhibits pericyte outgrowth in an aortic ring assay and pericyte migration towards endothelial cells.

<p>(A) Photomicrographs of mouse aortic ring cultured in collagen for 9 days and incubated without specific treatment (Ctrl) or under 16K hPRL treatment (16K). Data representative of at least 2 independent experiments are shown. <i>Bar</i>, 1 mm. (B) Quantification of migrating cell distribution around the aortic ring performed by computerized image analysis. Cell distribution is defined as the number of intersections between spreading cells and a grid of concentric rings, plotted as a function of the distance to the aortic fragment. Each curve is a mean of the cell network distribution obtained by averaging at least 5 individual distributions generated for each experimental condition. (C) Photomicrographs of a mouse aortic ring cultured in collagen for 9 days without specific treatment (Ctrl) or in the presence of 100 nM 16K hPRL. The rings were fixed for subsequent immunostaining: anti-Isolectin B4 Ab (IB4: green) identifying EC, anti-NG2 proteoglycan Ab (NG2: red) identifying PC/SMC. Nuclei were colored with DAPI (blue). Data representative of at least 2 independent experiments are shown. <i>Bar</i>, 100 µm. (D) HBVP migration towards HUVEC was assessed in an under-agarose migration coculture assay. HUVEC were treated for 72 h with 100 nM 16K hPRL (16K). d = migration distance between the beginning (solid line) and the end of the migration (dotted line). *, P<0.05.</p

Hypothetical model of 16K hPRL effects on Notch signaling in endothelial/pericyte interactions.

<p>When endothelial cells are cultured with pericytes, Dll4/Notch4 pathway is activated. 16K hPRL treatment (6 h) alters Dll4/Notch4 signaling by decreasing <i>DLL4</i>, <i>NOTCH4</i>, <i>HEY2</i> and <i>EPHRINB2</i> transcripts in endothelial cells. After 16 h of endothelial cell-pericyte cocultures, Notch3 and Dll1 are increased respectively in pericytes and endothelial cells. 16K hPRL inhibits the Dll1/Notch3 signaling by reducing <i>DLL1</i> transcripts in endothelial cells and <i>NOTCH3</i> and <i>aSMA</i> transcripts in pericytes.</p

Reduced pericyte coverage in B16F10 tumor vessels after 16K-Ad treatment.

<p>(A) Representative photographs of 80 µm thick tumor sections from 16K-Ad- and Null-Ad-inoculated mice, double-stained for the endothelial cell marker CD31 (green) and the mural cell markers α-SMA, desmin, and NG2 (red). (B) Computer-assisted image analysis was used to quantify pericyte coverage. The area occupied by vessels and the covered area were estimated. Coverage was calculated by dividing the latter area by the former. The data represent, for each set of conditions, the mean ratio ± SEM calculated for all the fields of all tumor sections (one section per tumor). We analyzed 10 tumors of Null-Ad-treated and 8 tumors of 16K-Ad-treated mice. <i>Bar</i>, 100 µm. (C) Representative photographs of tracheal capillaries. Tracheal capillaries from 16K-Ad- and Null-Ad-inoculated mice were visualized by lectin-FITC staining (green) and pericytes were visualized by desmin immunostaining (red). <i>Bar</i>, 25 µm. (D) Computer-assisted image analysis was used to quantify pericyte density. (E) RNAs were extracted from tumors and the relative abundance ± SEM of each specified mRNA (encoding pdgf-b, Ang1, or Ang2) was assessed by qRT-PCR in 10 Null-Ad tumors and 8 16K-Ad tumors. (F) Western blot analysis of Dll4 and (G) EphrinB2 in different 16K-Ad-treated and control tumors. n.s., non significant. *, P<0.05. **, P<0.001. ***, P<0.0001.</p

Decreased expression of PDGFR-B in HBVP cocultured with HUVEC after 16K hPRL treatment.

<p>A. CD31 (endothelial cells) and α-SMA(pericytes) immunohistochemistry on HUVEC and HBVP cells before and after separation with MACS. (B-E) HUVEC pretreated for 30 min with 100 nM 16K hPRL (16K) were cultured for 16 h in the presence or absence of HBVP. The two cell populations were then separated prior to qRT-PCR analysis and subjected to qRT-PCR analysis to detect <i>ANG2</i>, <i>ANG1</i>, <i>PDGFB</i> and PDGFR-B transcripts. Data are presented as means ± SEM, n = 3 and are representative of at least two different experiments. n.s., non significant. *, P<0.05.</p

Design, Synthesis, and Biological Evaluation of Novel, Highly Active Soft ROCK Inhibitors

ROCK1 and ROCK2 play important roles in numerous cellular functions, including smooth muscle cell contraction, cell proliferation, adhesion, and migration. Consequently, ROCK inhibitors are of interest for treating multiple indications including cardiovascular diseases, inflammatory and autoimmune diseases, lung diseases, and eye diseases. However, systemic inhibition of ROCK is expected to result in significant side effects. Strategies allowing reduced systemic exposure are therefore of interest. In a continuing effort toward identification of ROCK inhibitors, we here report the design, synthesis, and evaluation of novel soft ROCK inhibitors displaying an ester function allowing their rapid inactivation in the systemic circulation. Those compounds display subnanomolar activity against ROCK and strong differences of functional activity between parent compounds and expected metabolites. The binding mode of a representative compound was determined experimentally in a single-crystal X-ray diffraction study. Enzymes responsible for inactivation of these compounds once they enter systemic circulation are also discussed