A BMP7 variant inhibits tumor angiogenesis in vitro and in vivo through direct modulation of endothelial cell biology

Bone morphogenetic proteins (BMPs), members of the TGF-\u3b2 superfamily, have numerous biological activities including control of growth, differentiation, and vascular development. Using an in vitro co-culture endothelial cord formation assay, we investigated the role of a BMP7 variant (BMP7v) in VEGF, bFGF, and tumor-driven angiogenesis. BMP7v treatment led to disruption of neo-endothelial cord formation and regression of existing VEGF and bFGF cords in vitro. Using a series of tumor cell models capable of driving angiogenesis in vitro, BMP7v treatment completely blocked cord formation. Pre-treatment of endothelial cells with BMP7v significantly reduced their cord forming ability, indicating a direct effect on endothelial cell function. BMP7v activated the canonical SMAD signaling pathway in endothelial cells but targeted gene knockdown using shRNA directed against SMAD4 suggests this pathway is not required to mediate the anti-angiogenic effect. In contrast to SMAD activation, BMP7v selectively decreased ERK and AKT activation, significantly decreased endothelial cell migration and down-regulated expression of critical RTKs involved in VEGF and FGF angiogenic signaling, VEGFR2 and FGFR1 respectively. Importantly, in an in vivo angiogenic plug assay that serves as a measurement of angiogenesis, BMP7v significantly decreased hemoglobin content indicating inhibition of neoangiogenesis. In addition, BMP7v significantly decreased angiogenesis in glioblastoma stem-like cell (GSLC) Matrigel plugs and significantly impaired in vivo growth of a GSLC xenograft with a concomitant reduction in microvessel density. These data support BMP7v as a potent anti-angiogenic molecule that is effective in the context of tumor angiogenesis

Can, Want and Try: Parents' Viewpoints Regarding the Participation of Their Child with an Acquired Brain Injury

BACKGROUND: Acquired brain injury (ABI) is a leading cause of permanent disability, currently affecting 20,000 Australian children. Community participation is essential for childhood development and enjoyment, yet children with ABI can often experience barriers to participation. The factors which act as barriers and facilitators to community participation for children with an ABI are not well understood. AIM: To identify the viewpoints of parents of children with an ABI, regarding the barriers and facilitators most pertinent to community participation for their child. METHODS: Using Q-method, 41 parents of children with moderate/severe ABI sorted 37 statements regarding barriers and facilitators to community participation. Factor analysis identified three viewpoints. RESULTS: This study identified three distinct viewpoints, with the perceived ability to participate decreasing with a stepwise trend from parents who felt their child and family "can" participate in viewpoint one, to "want" in viewpoint two and "try" in viewpoint three. CONCLUSIONS: Findings indicated good participation outcomes for most children and families, however some families who were motivated to participate experienced significant barriers. The most significant facilitators included child motivation, supportive relationships from immediate family and friends, and supportive community attitudes. The lack of supportive relationships and attitudes was perceived as a fundamental barrier to community participation. SIGNIFICANCE: This research begins to address the paucity of information regarding those factors that impact upon the participation of children with an ABI in Australia. Findings have implications for therapists, service providers and community organisations

Discovery of a new azole resistance mechanism in Aspergillus fumigatus through whole genome sequencing and sexual crossing.

Contains fulltext : 110932.pdf (publisher's version ) (Open Access

BMP7v reduced vascularization <i>in vivo</i>.

<p>(A) Assessment of vascularity with immunofluorescence microscopy in Matrigel implants containing either GSLC1 or GSLC28 GFP expressing tumor cells (green) from control (CNTR) or BMP7v treated mice. The peripheral and central regions of implants were assessed for CD31 (red) and α-SMA (blue). (B) Graphs represent mean vessel density (number of vessels per microscopic field), total vessel area, and relative vessel area (total vessel area/area of microscopic field) from one experiment, and asterisks denote statistically significant (*, <i>p<</i>0.05) differences compared to controls. (C) Mice developing subcutaneous xenografts in the range of 9–13 mm entered the treatment schedule (vehicle n, 5; BMP7v 1 μg, n, 7; BMP7v 0.1 μg, n, 4). Time point “0” corresponds to the beginning of treatment. Values are expressed as means ± SEM, and arrows depict compound treatment. BMP7v was well tolerated with no apparent loss of body weight or overt signs of toxicity. In vehicle injected mice, the tumors progressively increased their diameter from 9.9 + 0.74 mm (mean + SEM) at Week 1 to 21 + 1.6 mm at Week 5. Statistically significant (<i>p</i><0.05) decreases in tumor size were observed at the 4 and 5 week measurements with 0.1 μg BMP7v treatment and (<i>p</i><0.01) at the 2, 3, 4, and 5 week timepoints with 1 μg BMP7v compared to vehicle controls (CNTR). (D) Analysis of CD31 immunostaining from GSLC efficacy study tumors as described in Methods.</p

BMP7v reduced VEGF and bFGF established cords.

<p>(A) The ADSC/ECFC co-culture was unstimulated (basal) or stimulated with 10 ng/ml VEGF or bFGF for 96 hours prior to treatment with PBS or 100 ng/ml BMP7v for 72 hours and immunohistochemistry for CD31 (green), α-smooth muscle actin (red), and Hoechst 33342 to stain all nuclei (blue). Representative images (4X magnification) from three independent experiments are shown, and graphs represent mean connected tube area ± SEM after normalization to growth factor-induced cord values; asterisks denote statistically significant (**, <i>p<</i>0.01) differences compared to PBS controls. (B) The ADSC/ECFC co-culture was unstimulated (basal) or stimulated with 10 ng/ml bFGF for 96 hours prior to treatment with PBS or 100 ng/ml BMP7v for 72 hours and immunohistochemistry for CD31 (green), nidogen or collagen IV (red) and Hoechst 33342 to stain all nuclei (blue). Representative images (5X magnification) are shown.</p

BMP7v reduced VEGFR2 and FGFR1 expression in endothelial cells.

<p>(A) Whole cell protein extracts were isolated following 15 or 60 minute PBS or 100 ng/ml BMP7v treatment and subjected to Western blot analysis using antiserum directed against phospho-SMAD1,5,8 (pSMAD1,5,8), phospho-ERK1/2 (pERK1/2), ERK1/2, and β-actin as a loading control. Results shown are representative of three independent experiments. (B) ECFCs were treated with PBS or 2nM BMP7v or BMP4 in ADSC conditioned defined co-culture media for the times indicated. Whole cell protein extracts were isolated following treatment and subjected to Western blot analysis using antiserum directed against phospho-CRAF (pCRAF), phospho-MEK1/2 (pMEK1/2), phospho-ERK1/2 (pERK1/2), and β-actin as a loading control. (C) Whole cell protein extracts were isolated from ECFCs following 72 hours of PBS or 100 ng/ml BMP7v treatment and subjected to Western blot analysis using antiserum directed against VEGFR2, FGFR1, and β-actin as a loading control. Graph represents mean densitometry ± SEM from three independent experiments, and asterisks denote statistically significant (***, <i>p<</i>0.001) differences compared to PBS controls. (D) The ADSC/ECFC co-culture was unstimulated (basal) or stimulated with 10 ng/ml VEGF or bFGF for 96 hours prior to treatment with PBS or 100 ng/ml BMP7v for 72 hours prior to immunohistochemistry for VEGFR2 (VEGF) or FGFR1 (bFGF) (red) and Hoechst 33342 to stain all nuclei (blue). Representative images (5X magnification) are shown. Upon BMP7v treatment, mean staining intensity for VEGFR2 and FGFR1 was statistically decreased (***, <i>p<</i>0.001) compared to PBS controls.</p

BMP7v inhibited cord formation in a SMAD4-independent manner.

<p>(A) Whole cell protein extracts were isolated from ECFCs following non-target (NT) or SMAD4 shRNA treatment and stable selection with puromycin. Extracts were subjected to Western blot analysis using antiserum directed against SMAD4 and β-actin as a loading control. Graph represents mean densitometry ± SEM from three independent experiments, and asterisks denote statistically significant (***, <i>p<</i>0.001) differences compared to PBS controls. (B) ECFCs were treated with non-target (NT) or SMAD4 shRNA followed by stable selection prior to plating into the ADSC/ECFC co-culture. The co-culture was stimulated with 10 ng/ml VEGF or bFGF simultaneously with PBS or 100 ng/ml BMP7v for 72 hours prior to immunohistochemistry for CD31 (green), α-smooth muscle actin (red), and Hoechst 33342 to stain all nuclei (blue). Representative images (5X magnification) are shown, graphs represent mean connected tube area ± SEM after basal cord formation data was subtracted from three independent experiments, and asterisks denote statistically significant (***, <i>p<</i>0.001) differences compared to PBS controls.</p