Orthopaedic Research Laboratory Alumni Council

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Board of Directors

Richard Debski, Ph.D. President
Caroline Wang, M.S. Secretary
Jamie Pfaeffle, M.D., Ph.D. Treasurer
Doug Boardman, M.D.
Thay Lee, Ph.D.
Patrick McMahon, M.D.
Karen Ohland, M.S.
Christos Papgeorgiou, M.D.
Masataka Sakane, M.D.
Sven Scheffler, M.D.
Jennifer Wayne, Ph.D.

2003 Mrs. Ho-Tung Cheong Grant Recipient

Laxminarayanan Krishnan
University of Utah

Gene Expression Pattern in an In Vitro Model of Angiogenesis

SUMMARY

The original proposal aimed at understanding the interactions between angiogenesis and tissue mechanical properties, based on an in vitro 3-D model of angiogenesis consisting of rat microvessels embedded in Type-1 collagen matrix. Post submission, the scope of the work was narrowed, in consultation with my advisor, to studying the gene expression pattern in our in vitro model of angiogenesis in the absence of external loading.   This establishes a baseline expression pattern of gene expression for our model. This expression pattern will provide useful information about molecular events in the constructs concomitant with the observed mechanical changes (1) in the absence of external mechanical loading.   Furthermore, we propose to compare this expression pattern with the expression levels in similar mechanically conditioned gels.

INTRODUCTION

Angiogenesis, the formation and growth of new vascular elements from existing ones, involves the stimulation of quiescent endothelial cells to exhibit migratory and proliferative phenotypes. Angiogenesis is regulated by a gamut of factors including chemotaxis, haptotaxis, cell-matrix mechanical forces, and mechanical loading via the extracellular matrix (ECM). Sprouting endothelial cells degrade their basement membrane and the surrounding ECM by MMP activity (2,3) , produce ECM proteins, and form contacts with and migrate along extracellular matrix components (4) , depositing a new basement membrane to form a patent, perfusion-capable capillary. Regulation of angiogenesis is a dynamic process influenced by the physico-chemical environment (2) . Little is known regarding the interaction of angiogenic microvessels with the ECM. In our research, we use a novel 3 D in vitro model of angiogenesis to isolate and investigate such influences. Our aim here was to quantify the changes in the expression profile of genes associated with angiogenesis in the absence of any external mechanical loading.

METHODS

A well established and characterized model of angiogenesis (5,6) was used in the in vitro studies. Microvessel fragments were isolated from epididymal fat pads of Sprague Dawley rats by partial collagenase digestion and sequential filtration through 500?m and 30?m nylon membranes. These fragments, consisting of arterioles, venules and capillaries, were seeded into collagen type I gels at a density of 15,000 frags/ml. Gels were polymerized in custom culture chambers and incubated at 37 degrees C, 95% humidity. Angiogenesis began predictably on the 4th day of culture and progressed to form a dense capillary network by the 10th day. The cultures were harvested for RNA extraction after the determined period (1, 6 or 10 days). Six gels for each of these time points were collected corresponding to time points of earlier mechanical testing.   Microvessel fragments equivalent to that needed for 6 cultures were used as Day 0 controls.   RNA was extracted using a Trizol based protocol (7) . RNA was assessed for purity and concentration, treated with DNAse, concentrated and finally reverse transcribed to cDNA. The cDNA was then quantified and an optimum minimum concentration necessary for our polymerase chain reactions (PCR) was determined (5ng/reaction).

PCR and Data Analysis

Expression levels of twenty two different gene targets which included housekeeping genes (GAPDH, HPRT, YWHZ, Dynactin2, UBC); Matrix metalloproteinases (MMP2, 9, 13, 14); Tissue inhibitors of metalloproteinases (TIMP1, 2); Collagens (Collagen 1, 3, 8); Growth factors (VEGF, FGF, PDGF, BMP1, VEGFR); Cell- matrix interaction genes ( Decorin, Fibronectin, Hyaluronic acid synthase).   10microliter reactions were run in triplicate using UDG Platinum Taq Supermix with SYBR green chemistry. All samples for a single target were evaluated in a run. The best housekeeping genes were determined by pairwise comparison procedures using GeNorm (8) and appropriate normalizations performed using a 'normalization factor' for each gene. Expression of each gene over the period of culture was examined to determine a baseline expression pattern of these genes. Mean expression levels at the four time points were compared to give a baseline expression pattern of that gene in the absence of any external loading. Multiple one-way ANOVAs were used to examine the differences for significance (alpha =0.05).   A preliminary analysis of our data is presented here.   We are currently examining error propagation methods in PCR in detail and their applicability to our data.

RESULTS

Matrix metalloproteinases showed significant upregulation with culture time. MMP2 levels were significantly upregulated through the culture period. MMP9 and MMP13 showed significant upregulation from Day 0 to Day 6. They did not show a significant change in expression between Day6 and Day 10. MMP14- a membrane bound MMP and TIMPs showed no significant differences with time (Figure 1).   Fibronectin expression did not differ significantly between Day0 & Day1 or Day6 and Day10. However, there was a significant increase in expression level between these two plateaus. Similarly, Decorin and Tenascin C showed an increase in expression between Day1 and Day6, with no change in expression between Day6 and Day10 (Figure 2).

DISCUSSION

The Collagen I matrix used in this system is formed by reassociation of collagen fibrils and differs from fibrillar collagen. The increase in levels of MMP2 and MMP9, 13 could be the primary cause of fall in construct stiffness observed in our mechanical tests (1). The decrease in dynamic stiffness of vascularized constructs with time can thus be explained by matrix degradation. We have noted an increase in the expression of cell matrix interaction elements like Fibronectin and Tenascin C, but not a significant change in the expression levels of growth factors. The early angiogenic process of sprout and network formation may be influenced more by cell matrix interaction elements than growth factors.  The late increase in Fibronectin and Tenascin C and a concomitant early spike of PDGF expression may mirror the actual proliferative and migratory phases of angiogenesis in the absence of modulation by other cells (fibroblasts, PMN). Decorin influences the association of collagen fibrils. Tenascin C is a glycoprotein regulating wound healing by limiting the amount of matrix deposition and also plays a adhesion modulating role (9). The relatively constant levels of collagen expression may reflect an early and prolonged stimulatory phase for ECM deposition. Similar explanation can be sought for growth factors and TIMPs, with upregulation of growth factors and downregulation of TIMPS beginning hours after stimuli or loss of mechanical preload. We are currently quantifying the MMP activity in these cultures and propose to relate them to the amount of gene upregulation observed and changes in matrix mechanical properties.

STATEMENT OF EXPENSES

The ORLAC summer research grant was beneficial in defraying partial costs of this project. Major section of the budgetary allowance was devoted to procuring animals for the study and associated molecular biology reagents for gene expression analysis. The budget however allowed us to procure a gel electrophoresis system, a standard water bath and pH meter. The gel electrophoresis system is equipped with an illumination system and has the novel application of real time tracking of migrating nucleotide fragments using SYBR green dye instead of an Ethidium bromide- Ultraviolet light system.

REFERENCES

  1. Krishnan, L., et al., Angiogenesis alters the material properties of the extracellular matrix.   Transactions of the Orthopaedic Research Society, 2003.
  2. Haas, T.L., S.J. Davis, and J.A. Madri, Three-dimensional type I collagen lattices induce coordinate expression of matrix metalloproteinases MT1-MMP and MMP-2 in microvascular endothelial cells.   J Biol Chem, 1998.   273 (6): p. 3604-10.
  3. Brown, M.D. and O. Hudlicka, Modulation of physiological angiogenesis in skeletal muscle by mechanical forces: Involvement of VEGF and metalloproteinases.   Angiogenesis, 2003.   6 : p. 1- 14.
  4. Vernon, R.B., and E.H. Sage, A novel, quantitative model for study of endothelial cell migration and sprout formation within three-dimensional collagen matrices.   Microvascular Res, 1999.   57 (2): p. 118-33.
  5. Hoying, J.B., C.A. Boswell, and S.K. Williams, Angiogenic potential of microvessel fragments established in three-dimensional collagen gels.   In Vitro Cell Dev Biol, 1996.   32 : p. 402-419.
  6. Hoying, J.B., et al., Rapid Perfusion of a Prevascularized Tissue Construct Following Implantation.   Laboratory Investigation, 2002.   In Review .
  7. Reno, C., et al., Rapid isolation of total RNA from small samples of hypocellular, dense connective tissues.   Biotechniques, 1997.   22 (6): p. 1082-6.
  8. Vandesompele, J., et al., Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes.   Genome Biol, 2002.   3 (7): p. RESEARCH0034.
  9. Midwood, K.S., and J.E. Schwarzbauer, Tenascin C modulates matrix contraction via focal adhesion kinase- and rho-mediated signaling pathways.   Molecular Biology of the Cell, 2002.   13 : p. 3601-3613.

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