Implantation of oxygen enhanced, three-dimensional microporous L-PLA polymers: a reproducible porcine model of chronic total coronary occlusion.

We have hypothesized that oxygen enhanced three-dimensional microporous poly-L-lactic acid (L-PLA) bioabsorbable polymer constructs could be implanted to produce a subacute occlusion in a porcine coronary artery, forming a thrombofibrotic occlusion containing microvascular channels. Chronic total occlusion (CTO) is increasingly prevalent in patients who present for percutaneous interventions. No reproducible animal coronary model simulating human CTOs has previously been developed. Swine coronary arteries were cannulated and a microporous L-PLA polymer pledget was advanced into a preselected segment of coronary. The coronary arteries were angiographically re-imaged at day 3, day 10, and day 28, to document the presence or absence of an occlusion. Histopathology was also performed at each time point to evaluate the lesion characteristics. A novel three-dimensional L-PLA microporous polymer construct, when implanted into porcine coronary arteries, reproducibly results in the development of a CTO at day 3. The histopathology in this porcine coronary model of CTO at day 28 closely mimics human coronary CTO, including the presence of microvascular channels and dense collagen and elastic tissue in the occlusion.

Proliferation and beta-tubulin for human aortic endothelial cells within gas-plasma scaffolds.

PURPOSE: We determined if human aortic endothelial cells (HAEC) enhanced proliferative and angiogenic phenotypes within gas-plasma treated bioresorbable D,L-polylactic acid (D,L-PLA) three-dimensional scaffolds. METHOD: 6 x 10(3) HAEC (N=120) were incubated for 6, 12 or 18 days within either non-treated control or treated scaffolds. Before removing media, unstained wells were observed for apparent cell densities. Quantitative colorimetric WST-1 mitochondrial assays were determined for pooled conditioned media from both HAEC attached to wells and their respective HAEC-containing scaffolds. Fixed HAEC in scaffolds were examined using non-quantitative laser confocal microcopy with FITC-conjugated consensus, Types-I/II or Type-III beta-tubulin. RESULTS: WST-1 indicated that significantly (p<0.05) less mitochondria were on cell culture plates than inside scaffolds but for different reasons. For example, a 12-18 days comparison between WST-1 and beta-tubulin indicated that wells decreased because of overgrowth apotosis; whereas, mitochondrial activity inside treated scaffolds decreased with increased tubulogenesis. Observed with consensus and Type-I/II beta-tubulin, HAEC-treated scaffolds exhibited increased cell-cell interconnections and angiogenic cords undergoing tubulogenesis to form vessels with central lumens as well as increased Type-III beta-tubulin, predominantly in cells of smaller surface areas. Moreover, beta-tubulin inside HAEC-treated scaffolds appeared in discrete cytoskeletal and podial regions; yet, beta-tubulin for HAEC-control scaffolds was located in more diffuse cytoplasmic regions especially at 18 days. CONCLUSIONS: HAEC-treated scaffolds undergo increased migration, proliferation, beta-tubulin expression and quiescent cord formation. HAEC in scaffolds represent a potential model to study mechanisms for vascular cord progression into tubes. WST-1 does not represent accurate cell densities in three-dimensional scaffold matrices.

High glucose induced nuclear factor kappa B mediated inhibition of endothelial cell migration.

Delayed wound healing and accelerated atherosclerosis are common vascular complications of diabetes mellitus. Although elevated blood glucose level is the major contributing factor, mechanisms that mediate these complications are not clearly understood. In the present study, we have demonstrated that elevated glucose inhibits endothelial cell migration, thereby delaying wound healing. Our results clearly indicated that high glucose (10 or 30 mM) induced activation of nuclear factor kappa B (NF-kappaB) inhibited endothelial cell migration (P<0.05). High glucose induced NF-kappaB DNA binding activity may mediate this inhibition of migration by regulating intracellular nitric oxide. In vitro wound healing model in human aortic endothelial cells (HAEC) were used to evaluate cell migration under the influence of high glucose. The migration inhibited by high glucose was restored by NF-kappaB inhibitors (including E3-4-methylphenyl sulfonyl-2-propenenitrile, N-tosyl-Lys-chloromethylketone (TLCK), or over-expression of inhibitor subunit of kappaB) and endothelial nitric oxide synthase inhibitors (N-methyl-L-arginine (L-NMMA); and Nomega-nitro-L-arginine methyl ester (L-NAME)). Furthermore, NF-kappaB inhibitors attenuated high glucose induced eNOS expression and intracellular nitric oxide (NO) production. Cytoskeletal immunofluorescence staining confirmed differences in actin distribution in HAEC incubated in high glucose in the presence or absence of NF-kappaB and NO inhibitors, explaining the differences observed in migration. In summary, our results for the first time suggest therapeutic strategies involving inhibition of NF-kappaB activation induced by high glucose, which may improve wound healing and help avoid some of the vascular complications of diabetes.

Angiogenic bFGF expression from gas-plasma treated scaffolds.

PURPOSE: In vivo experiments indicate that gas-plasma-treated D,L-polylactide polymers expressing basic fibroblast growth factor (bFGF) exhibit enhanced angiogenesis. bFGF is not a single entity, but it is instead a family of isoforms. Consequently, we sought to determine which bFGF isoforms and levels initiate angiogenesis in nude mice peritoneums. METHODS: Cytoplasmic and nuclear bFGF were characterized for nude mice peritoneums incubated with nontreated scaffolds containing HAEC (CW), its respective polymer-only scaffolds (Cp) and gas-plasma treated scaffolds with HAEC (TW) and without cells (Tp). NuPAGE electrophoresis and WesternBreeze Chemiluminescent kits were used to analyze relative bFGF densities and molecular weights. VEGF was quantified using ImageJ. RESULTS: bFGF bands were located at molecular weights of 24, 48, 58, 72 and 80 kDa, depending on whether they were from cytoplasms or nuclei. At 12, 24 and 72 days, 58-kDa bFGF bands were observed from nuclei of TW and Tp, 80-kDa bFGF bands were only observed in cytoplasmic fractions < or = 24 days. Total cytoplasmic and nuclear bFGF intensities increased from 12 to 24 days, then declined by 72 days. CONCLUSIONS: (1) Gas-plasma treated scaffolds up-regulate bFGF isoforms. (2) bFGF was expressed in the nuclei; however, 80-kDa bFGF was seen only in cytoplasms.

VEGF analysis induced by endothelialized gas-plasma treated D,L-PLA scaffolds.

PURPOSE: Vascular endothelial growth factor (VEGF) isoforms play different roles in the temporal sprouting of endothelial-lined vessels in a nude mouse peritoneal model as cells respond to nontreated control and gas-plasma-treated bioresorbable poly-D,L-lactide acid 3D scaffolds with human aortic endothelial cells (HAEC). METHODS AND MATERIALS: Nude mice peritoneums were incubated with HAEC (CW = control; TW = gas-plasma treated) or polymer scaffolds (Cp = control; Tp = treated) for 12, 24 and 72 days. Cytoplasmic and nuclear protein fractions were isolated using NER, electrophoresized using NuPAGE-MES and analyzed by WesternBreeze Chemiluminescent. RESULTS: Prominent VEGF bands included 28, 45 and 62 kDa; 52-kDa VEGF observed in cytoplasmic TW fractions contributed about 18.6% at 12 days, 20.0% at 24 days and 13.1% at 72 days of the total VEGF signal. Yet, it was only noted in CW at 72 days where it accounted for 6.9%. A unique 32-kDa band appeared in both Cp (24.6%) and Tp (18.3%). Significant differences between band densities occurred for cytoplasmic nuclear CW24- TW24 (P = .022), CW72-TW72 (P = .011) and, also, cytoplasmic Cp24-Tp24 (P = .038). CONCLUSIONS: The temporal and spatial organization of the TW isoforms results in more angiogenesis.