Targeted Nano-Therapeutices for the Reversal of Calcification and ECM Degradation in Abdominal Aortic Aneurysms

Abdominal aortic aneurysm (AAA) is focal ballooning and dilation of abdominal aorta. AAAs are the 13th primary cause of death in the US, taking the lives of approximately 15,000 Americans each year 1. The best prevention of AAA is early detection when the AAA is smaller than 3 cm. The only current treatment option for well-developed AAA is surgical repair of the aneurysmal vessel. Since the enactment of the Screening Abdominal Aortic Aneurysms Very Efficiently Act (SAAVE) act, patients with Medicare are covered for one-time ultrasound imaging for aneurysm, which allows smaller scale AAAs to be discovered. Unfortunately, after this initial detection there is no currently known treatment to slow the growth of these aneurysms. Monitoring can be continued through the use of ultrasound imaging or plain film radiography, which shows calcification related to the AAA, but there is no effective way to show a full picture of focal wall weakening 2. When the AAA has a diameter greater than 5.5 cm, elective surgery is typically performed as the risk of surgery is less than the risk of a ruptured AAA. Excessive activity of metalloproteinases (MMPs) has been associated with aortic elastin damage and degeneratio3. Also AAA is often associated with calcification, which increases the risk of AAA rupture 4. Based on this knowledge, we hypothesized that combined treatment MMP inhibitors locally to stop the degradation of elastin, and pentagalloyl glucose (PGG) to regenerate lost elastin can be an effective treatment option for early to middle stage aneurysms in order to prevent disease progression. Furthermore, we hypothesized that calcification associated with well-developed AAA can be removed first with ethylene diamime tetraacetic acid (EDTA), a well-known metal ion chelator and then degraded elastin can be regenerated with PGG. The overall goal of this study was to develop a minimally invasive, non-toxic, targeted vascular drug delivery system that both prevents elastin degradation and aids elastin regeneration, thereby acting as a multi-functional treatment option for elasto-degenerative vascular diseases including AAAs. In order to achieve this goal, we used nanoparticles (NPs) developed in our laboratory to target degraded elastin by conjugating elastin antibody that specifically recognizes only degraded elastin. First, using calcium chloride-induced AAA rat model, we show targeted delivery of such nanoparticles loaded MMP inhibitor (BB-94) lead to suppression of MMP activity in abdominal aortic aneurysms and prevented aneurysmal expansion. Next, we demonstrated that nanoparticles can be loaded with PGG. In AAA model in rats, we show PGG can be delivered at the site of AAA by targeted NPs. Such PGG delivery inhibited elastin degradation and lead to suppression of AAA. Finally, we tested whether moderate size calcified aneurysms could be reversed by dual therapy. We created moderate size AAA in rats by calcium chloride injury, and then first delivered EDTA loaded NPs systemically to remove calcification followed by delivery of PGG loaded NPs. Only dual therapy showed reversal of calcification in the aorta as well as reversal of AAA and regeneration of elastic lamina. NPs with EDTA alone or blank NPs did not cause regeneration of elastic lamina in the aorta

Gene Expression Profiling of Human Adipose Tissue Stem Cells during 2D versus 3D Adipogenesis

Much of the current understanding on molecular and cellular events of adipose developmental biology comes from monolayer cell culture models using preadipocyte cell lines, although in vivo adipose tissue consists of a much more complex three-dimensional microenvironment of diverse cell types, extracellular network, and tissue-specific morphological and functional features. Added to this fact, the preadipocytes, on which the adipogenesis mechanisms are mostly explored, possess some serious limitations (e.g., time of initial subculture and adipogenic differentiation time), which, perhaps, can efficiently be replaced with progenitor cells such as adipose tissue-derived stem cells (ASCs). With the objective of developing a better in vitro model for adipose developmental biology, this project involves gene expression profiling of human ASCs (hASCs) during their differentiation to adipocytes in a 2D versus 3D culture model. This transcriptional-level analysis revealed that gene expression patterns of adipogenesis-induced hASCs in a 3D self-assembled polypeptide hydrogel are relatively different from the 2D monolayered cells on plastic hard substrate. Moreover, analysis of adipogenic lineage progression 9 days after adipogenic induction shows earlier differentiation of hASCs in 2D over their 3D counterparts. However, differentiation in 2D shows some unexpected behavior in terms of gene expression, which does not seem to be related to adipogenic lineage specification. Since hASCs are already being used in clinical trials due to their therapeutic potential, it is important to have a clear understanding of the molecular mechanisms in an in vivo model microenvironment like the one presented here

Calcium Phosphate/Etidronate Disodium Biocement: Etidronate, Retarder or Accelerator

Bisphosphonate release from calcium phosphate cement has been investigated. We hypothesized that local delivery of bisphosphonate from the calcium phosphate cement improves the mechanical properties. Different samples with different concentration of Etidronate disodium have been made and analyzed. We observed a dual behavior from Etidronate in retarding and accelerating the setting of calcium phosphate cement in low and high concentration respectively. After soaking samples in simulated body fluid, an optimum concentration of Etidronate disodium was added to the calcium phosphate paste in order to achieve the best mechanical properties. Scanning electron microscopy (SEM) showed the formation of hydroxyapatite crystals. X-ray diffraction (XRD) analysis was used to determine hydroxyapatite peaks on the surface of the bio-cement, which confirms the hydroxyapatite formation

Mutability of druggable kinases and pro-inflammatory cytokines by their proximity to telomeres and A+T content.

Mutations of protein kinases and cytokines are common and can cause cancer and other diseases. However, our understanding of the mutability in these genes remains rudimentary. Therefore, given previously known factors which are associated with high mutation rates, we analyzed how many genes encoding druggable kinases match (i) proximity to telomeres or (ii) high A+T content. We extracted this genomic information using the National Institute of Health Genome Data Viewer. First, among 129 druggable human kinase genes studied, 106 genes satisfied either factors (i) or (ii), resulting in an 82% match. Moreover, a similar 85% match rate was found in 73 genes encoding pro-inflammatory cytokines of multisystem inflammatory syndrome in children. Based on these promising matching rates, we further compared these two factors utilizing 20 de novo mutations of mice exposed to space-like ionizing radiation, in order to determine if these seemingly random mutations were similarly predictable with this strategy. However, only 10 of these 20 murine genetic loci met (i) or (ii), leading to only a 50% match. When compared with the mechanisms of top-selling FDA approved drugs, this data suggests that matching rate analysis on druggable targets is feasible to systematically prioritize the relative mutability-and therefore therapeutic potential-of the novel candidates