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Biglycan potentially regulates angiogenesis during fracture repair by altering expression and function of endostatin.

ABSTRACT: The small proteoglycan biglycan (Bgn) is highly expressed in the organic matrix of bone and plays a role in bone formation. Previous work implicated Bgn in vessel growth during bone healing [1]. By infusing barium sulfate (BaSO4) into WT and Bgn-deficient mice we discovered the positive effect of Bgn in modulating angiogenesis during fracture healing. Using micro-computed tomography angiography we found significant differences in the vessel size and volume among other parameters. To further understand the mechanistic basis for this, we explored the relationship between Bgn and the anti-angiogenic protein endostatin. Immunohistochemistry (IHC) showed co-localization of Bgn and endostatin in regions of bone formation, with increased endostatin staining in Bgn-KO compared to WT at 14days post-fracture. To further elucidate the relationship between Bgn and endostatin, an endothelial cell tube formation assay was used. This study showed that endothelial cells treated with endostatin had significantly decreased vessel length and vessel branches compared to untreated cells, while cells treated with endostatin and Bgn at a 1:1M ratio had vessel length and vessel branches comparable to untreated cells. This indicated that Bgn was able to mitigate the inhibitory effect of endostatin on endothelial cell growth. In summary, these results suggest that Bgn is needed for proper blood vessel formation during fracture healing, and one mechanism by which Bgn impacts angiogenesis is through inhibition of endostatin.

SUBMITTER: Myren M

PROVIDER: S-EPMC4875882 | biostudies-literature | 2016 May-Jul

REPOSITORIES: biostudies-literature

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Biglycan potentially regulates angiogenesis during fracture repair by altering expression and function of endostatin.

Myren Maja M Kirby David J DJ Noonan Megan L ML Maeda Azusa A Owens Rick T RT Ricard-Blum Sylvie S Kram Vardit V Kilts Tina M TM Young Marian F MF

Matrix biology : journal of the International Society for Matrix Biology 20160409

The small proteoglycan biglycan (Bgn) is highly expressed in the organic matrix of bone and plays a role in bone formation. Previous work implicated Bgn in vessel growth during bone healing [1]. By infusing barium sulfate (BaSO4) into WT and Bgn-deficient mice we discovered the positive effect of Bgn in modulating angiogenesis during fracture healing. Using micro-computed tomography angiography we found significant differences in the vessel size and volume among other parameters. To further unde ...[more]

PMID: 27072616

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Project description:Matrix proteoglycans such as biglycan (Bgn) dominate skeletal tissue and yet its exact role in regulating bone function is still unclear. In this paper we describe the potential role of (Bgn) in the fracture healing process. We hypothesized that Bgn could regulate fracture healing because of previous work showing that it can affect normal bone formation. To test this hypothesis, we created fractures in femurs of 6-week-old male wild type (WT or Bgn+/0) and Bgn-deficient (Bgn-KO or Bgn-/0) mice using a custom-made standardized fracture device, and analyzed the process of healing over time. The formation of a callus around the fracture site was observed at both 7 and 14 days post-fracture in WT and Bgn-deficient mice and immunohistochemistry revealed that Bgn was highly expressed in the fracture callus of WT mice, localizing within woven bone and cartilage. Micro-computed tomography (?CT) analysis of the region surrounding the fracture line showed that the Bgn-deficient mice had a smaller callus than WT mice. Histology of the same region also showed the presence of less cartilage and woven bone in the Bgn-deficient mice compared to WT mice. Picrosirius red staining of the callus visualized under polarized light showed that there was less fibrillar collagen in the Bgn-deficient mice, a finding confirmed by immunohistochemistry using antibodies to type I collagen. Interestingly, real time RT-PCR of the callus at 7 days post-fracture showed a significant decrease in relative vascular endothelial growth factor A (VEGF) gene expression by Bgn-deficient mice as compared to WT. Moreover, VEGF was shown to bind directly to Bgn through a solid-phase binding assay. The inability of Bgn to directly enhance VEGF-induced signaling suggests that Bgn has a unique role in regulating vessel formation, potentially related to VEGF storage or stabilization in the matrix. Taken together, these results suggest that Bgn has a regulatory role in the process of bone formation during fracture healing, and further, that reduced angiogenesis could be the molecular basis.

Project description:BACKGROUND:Despite its functional importance in various fundamental bioprocesses, studies of N6-methyladenosine (m6A) in the heart are lacking. Here, we show that the FTO (fat mass and obesity-associated protein), an m6A demethylase, plays a critical role in cardiac contractile function during homeostasis, remodeling, and regeneration. METHODS:We used clinical human samples, preclinical pig and mouse models, and primary cardiomyocyte cell cultures to study the functional role of m6A and FTO in the heart and in cardiomyocytes. We modulated expression of FTO by using adeno-associated virus serotype 9 (in vivo), adenovirus (both in vivo and in vitro), and small interfering RNAs (in vitro) to study its function in regulating cardiomyocyte m6A, calcium dynamics and contractility, and cardiac function postischemia. We performed methylated (m6A) RNA immunoprecipitation sequencing to map transcriptome-wide m6A, and methylated (m6A) RNA immunoprecipitation quantitative polymerase chain reaction assays to map and validate m6A in individual transcripts, in healthy and failing hearts, and in myocytes. RESULTS:We discovered that FTO has decreased expression in failing mammalian hearts and hypoxic cardiomyocytes, thereby increasing m6A in RNA and decreasing cardiomyocyte contractile function. Improving expression of FTO in failing mouse hearts attenuated the ischemia-induced increase in m6A and decrease in cardiac contractile function. This is performed by the demethylation activity of FTO, which selectively demethylates cardiac contractile transcripts, thus preventing their degradation and improving their protein expression under ischemia. In addition, we demonstrate that FTO overexpression in mouse models of myocardial infarction decreased fibrosis and enhanced angiogenesis. CONCLUSIONS:Collectively, our study demonstrates the functional importance of the FTO-dependent cardiac m6A methylome in cardiac contraction during heart failure and provides a novel mechanistic insight into the therapeutic mechanisms of FTO.

Dataset Information

Biglycan potentially regulates angiogenesis during fracture repair by altering expression and function of endostatin.

Publications

Biglycan potentially regulates angiogenesis during fracture repair by altering expression and function of endostatin.

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