Human Osteogenic Adipose-Derived Progenitor Cells as a Source of Vascular Endothelial Growth Factor

Cell-based therapies have shown particular promise in several difficult-to-treat conditions and diseases [5-7]. Human adipose-derived stem cells (ASCs) hold unique qualities for use in regenerative medicine. A well-known benefit of ASCs lies in their unique ability to differentiate along with several mesodermal and endothelial lineages as human adipose-derived progenitor cells (APCs) [8,9]. APCs may directly repair, replace, or regenerate multiple cell types in the cardiac myocardium [10-13]. In addition to the regenerative benefit of multi-differentiation, ASCs also secrete several cytokines that promote angiogenesis. One of the most well studied angiogenic cytokine is vascular endothelial growth factor (VEGF) [8,9,14,15]. VEGF is involved in several angiogenic functions, to include endothelial cell migration, mitogenesis, vascular sprouting, and vascular tube formation [16,17]. VEGF-A is the most active multi-tasking factor among the VEGF family of proangiogenic cytokines and is essential for cardiovascular development and function [18,19].


Abbreviations
. This is an index qualitatively descriptive study; no statistical analysis was pursued.

Method of SVF isolation
Fresh human waste lipoaspirate from informed and consented three healthy females were used for this University of Florida Jacksonville Institutional Review Board approved study (IRB# 201601520). Each of the three lipoaspirate samples was used to prospectively establish a cell line, from initial ASC expansion to differentiation and analysis. The lipoaspirate had been harvested using standard operative tumescent techniques using syringe liposuction. The tumescent solution included normal saline, lidocaine, and epinephrine. Shortly af-ter harvest, the fresh lipoaspirate was then processed using the "Reviticell Kit" (Reviticell, Inc., Jacksonville, FL) with its inherent 10-step process. In brief, shortly after the lipoaspirate was obtained, 70mls of unconcentrated lipoaspirate was measured out and subsequently underwent intersyringe transfer (between two 60 ml syringes provided as part of the Reviticell Kit) for five transfers. Then, 17. 5

Discussion
ARCserve an essential role in cardiac cell and tissue regeneration by offering both structural and hormonal regenerative capabilities. From a structural perspective, ASCs exist in an undifferentiated state, with the ability to self-renew and differentiate along several mesodermal APC lineages [8,9,[30][31][32][33][34][35].
Additionally, animal studies have shown that ASCs have the potential to differentiate in vivo into endothelial cells and cardiomyocytes [34,36,37]. While the maintenance and production of vessels rely on several growth factors and cytokines, VEGF is one of the most critical and well-studied angiogenic growth factors [38]. Hormonally, ASCs have been shown to improve blood flow in a mouse model of hind limb ischemia by secreting angiogenic growth factors, including VEGF [39].
While it appears counterintuitive to deliver osteogenic APCs to adult cardiac tissues damaged by ischemia, it is noteworthy to mention that bone morphogenetic proteins play a well-established role in both vascular growth [40] and cardiac development [41]. Heterotopic bone formation is also a consistent finding in valvular heart disease [42]. Up-regulating angiogenesis and down-regulating bone formation in the therapeutic use of osteogenic APCs for cardiovascular disease will be critical. The current study demonstrates that APCs secreted levels of VEGF-A 165 in a lineage-dependent manner and osteogenic APCs    This osteogenic progenitor sphere has been stained with Alizarin Red (for calcium) and DAB (for VEGF-A 165 ), 20x mag. The staining is comparatively lighter than with DAB alone and darker than Alizarin Red alone, diagnostic for an osteogenic progenitor sphere producing VEGF-A 165 .   [43,44] but, not osteoblast differentiation [45]. The latter finding suggests that enhanced VEGF-A secretion is not a sufficient determinant of bone formation by osteogenic APCs. In this current study, it may be intuitive to assume that a homogenous population of ASCs treated with an osteogenic differentiation medium may become VEGF-A 165 secreting osteogenic progenitor cells; indeed, the VEGF-A 165 -DAB immunostain in combination with the calcium-Alizarin Red stain proved this assumption correct.
The potency of APCs with respect to VEGF-A production may be compared to the known enzymatic activity of rVEGF 165 . The cell proliferation assay against which rVEGF activity is measured includes HUVEC stimulation [46] The median effective dose (ED 50 ) for this effect of Current Good Manufacturing Grade quality rVEGFis 1-6 ng/mL with a specific activity of approximately 1.7 x 10 3 U/µg (Cat# 293GMP, R&D Systems, Minneapolis, MN). As a comparative measure, 1.93 x 10 5 osteogenic APCs would be needed to achieve an ED 50 for HUVEC stimulation based on known activity of rVEGF 165 .In vivo modeling for the efficacy of cardiac performance of with secreted VEGF-A 165 , as elaborated by infused ASCs, will need to be compared against rVEGF-A 165 infusion in a head-to-head study.
Therapeutic in vivo tissue regeneration will require the classical components that ex vivo tissue engineering requires: matrix scaffolds, cells, and signaling molecules. However, a stark difference between in vivo and ex vivo tissue engineering will be the inherent more complex dynamics between these three entities as appreciated vivo. While the complete characterization of ASCs, much fewer APCs, is currently unknown, designing regenerative therapies that will leverage the original biological characteristics of these therapeutic cells will be mandatory. As can be seen from the three cell lines, each cell line produced differing quantities of VEGF-A 165 though each cell line was cultured in ostensibly same culture conditions. A limitation of this study is the small sample size (three cell lines) and certainly, further studies instituting a greater cell line quantity will be needed to better assess these current findings. Finally, while speculative, including ASCs as cellular constituents to the tissue-building process, may afford unintended benefits; for example, they may differ to fibroblasts to produce matrix proteins (such as collagen), they may differentiate to cells to stabilize the micro-milieu (such as in their anti-inflammatory abilities), and they may differ to cells to produce signaling cytokines (such as VEGF). However, what is not speculative is that we cannot live without them. Matching their original biological characteristics to targeted therapies will be essential in establishing much-needed healthcare solutions.