Embryonic periventricular endothelial cells demonstrate a unique pro-neurodevelopment and anti-inflammatory gene signature.

Brain embryonic periventricular endothelial cells (PVEC) crosstalk with neural progenitor cells (NPC) promoting mutual proliferation, formation of tubular-like structures in the former and maintenance of stemness in the latter. To better characterize this interaction, we conducted a comparative transcriptome analysis of mouse PVEC vs. adult brain endothelial cells (ABEC) in mono-culture or NPC co-culture. We identified > 6000 differentially expressed genes (DEG), regardless of culture condition. PVEC exhibited a 30-fold greater response to NPC than ABEC (411 vs. 13 DEG). Gene Ontology (GO) analysis of DEG that were higher or lower in PVEC vs. ABEC identified "Nervous system development" and "Response to Stress" as the top significantly different biological process, respectively. Enrichment in canonical pathways included HIF1A, FGF/stemness, WNT signaling, interferon signaling and complement. Solute carriers (SLC) and ABC transporters represented an important subset of DEG, underscoring PVEC's implication in blood-brain barrier formation and maintenance of nutrient-rich/non-toxic environment. Our work characterizes the gene signature of PVEC and their important partnership with NPC, underpinning their unique role in maintaining a healthy neurovascular niche, and in supporting brain development. This information may pave the way for additional studies to explore their therapeutic potential in neuro-degenerative diseases, such as Alzheimer's and Parkinson's disease.

Bidirectional crosstalk between periventricular endothelial cells and neural progenitor cells promotes the formation of a neurovascular unit.

Interactions between neural progenitor cells (NPC) and endothelial cells (EC) from adult vascular beds have been well explored previously. However, the factors and signaling mechanisms that regulate neurogenesis and angiogenesis are most prevalent during embryonic development. This study aimed to determine whether embryonic brain endothelial cells from the periventricular region (PVEC) present an advantage over adult brain EC in supporting NPC growth and differentiation. PVEC were isolated from E15 mouse brains, processed, and sorted with immunomagnetic beads using antibodies against CD31/PECAM. On immunofluorescence (IF) staining, nearly all cells were positive for EC markers CD31 and CD144/VE-Cadherin. In proliferation studies, NPC proliferation was highest in transwell co-culture with PVEC, approximately 2.3 fold increase compared to baseline versus 1.4 fold increase when co-cultured with adult brain endothelial cells (ABEC). These results correlated with the PVEC mediated delay in NPC differentiation, evidenced by high expression of progenitor marker Nestin evaluated by IF staining. Upon further characterization of PVEC in an angiogenesis assay measuring cord length, PVEC exhibited a high capacity to form cords in basal conditions compared to ABEC. This was enhanced in the presence of NPC, with both cell types displaying a preferential structural alignment resembling neurovascular networks. PVEC also expressed high Vegfa levels at baseline in comparison to NPC and ABEC. Vegfa levels increased when co-cultured with NPC. We demonstrate that PVEC and NPC co-cultures act synergistically to promote the formation of a neurovascular unit through dynamic and reciprocal communication. Our results suggest that PVEC/NPC could provide promising neuro-regenerative therapies for patients suffering brain injuries.

In vivo testing of an infection-resistant annuloplasty ring.

BACKGROUND: An infection-resistant surface incorporated into a prosthetic cardiac valve has great potential clinical applications. STUDY DESIGN: A sewing ring construct was created using ciprofloxacin-treated polyester. Then ciprofloxacin-treated and untreated constructs were implanted subcutaneously on the dorsum of rats and inoculated with Staphylococcus aureus. At 7, 14, and 30 days animals were sacrificed and the implants were retrieved. Each implant was assessed for frank purulence and gross tissue incorporation by a blinded observer. The implants were processed for conventional histology and examined by a blinded Pathologist. Ciprofloxacin-treated rings were also implanted in the absence of a bacterial challenge. At explantation, a maximal zone of inhibition, if present, was measured. Finally, ciprofloxacin was eluted with methanol from the explanted segments and the concentration of ciprofloxacin eluted was determined. RESULTS: Ciprofloxacin-treated sewing rings had greater gross tissue incorporation than untreated rings in the presence of a bacterial challenge (P=0.005). Ciprofloxacin-treated rings also had a lower incidence of frank purulence, but this did not reach statistical significance. After 14 days of implantation, ciprofloxacin treated rings had fewer neutrophils (P=0.018) and greater histological tissue incorporation (P=0.017) than untreated rings. The explanted ciprofloxacin-treated rings maintained a zone of inhibition of 3.0+/-1.0 mm after 1 day of implantation and 1.3+/-0.6 mm after 2 days. Ciprofloxacin could be eluted in significant quantities from the explanted rings after 7 days of implantation. CONCLUSIONS: Ciprofloxacin treated polyester can be incorporated into an annuloplasty ring construct that demonstrates excellent tissue incorporation and infection resistance. This study supports the use of this construct in the mitral position in a large animal model.

Development of an infection-resistant, bioactive wound dressing surface.

Trauma, whether caused by an accident or in an intentional manner, results in significant morbidity and mortality. The goal of this study was to develop a novel biomaterial surface in vitro and ex vivo that provides both localized infection resistance nd hemostatic properties. Our hypothesis is that a combination of specific surface characteristics can be successfully incorporated into a single biomaterial. Functional groups were created with woven Dacron (Cntrl) material via exposure to ethylenediamine (C-EDA). The antibiotic ciprofloxacin (Cipro) was then applied to the C-EDA material using pad/autoclave technique (C-EDA-AB) followed by surface immobilization of the coagulation cascade enzyme thrombin (C-EDA-AB-Thrombin). Antimicrobial activity by the C-EDA-AB surface persisted for 5 days compared with Cntrl and dipped controls, which lasted <1 h. C-EDA-AB-Thrombin surfaces had 2.6- and 105-fold greater surface thrombin activity compared with nonspecifically bound thrombin and Cipro-dyed surfaces, respectively. Surface thrombus formation ex vivo was evident after 1 min of exposure, with thrombus organization evident by 2.5 min. In contrast, C-EDA-AB and Cntrl segments showed only blood protein adsorption on the fibers. Thus, this study demonstrated that Cipro and thrombin can be simultaneously incorporated onto a biomaterial surface while maintaining their respective biological activities.

Temporal gene expression following prosthetic arterial grafting.

BACKGROUND: Following prosthetic arterial grafting, cytokines and growth factors released within the perianastomotic tissues stimulate smooth muscle cell proliferation and matrix production. While much in vitro work has characterized this response, little understanding exists regarding the sequential up- and down-regulation of genes following prosthetic arterial grafting. This study evaluates temporal gene expression at the distal anastomosis of prosthetic arterial grafts using microarray analysis. METHODS: Expanded polytetrafluoroethylene (ePTFE) carotid interposition grafts (n = 12) were surgically implanted into mongrel dogs. Distal anastomotic segments were harvested at 7, 14, 30, or 60 days. Contralateral carotid artery served as control. Total RNA was isolated from the anastomotic tissue and paired controls. Samples were probed with oligonucleotide microarrays consisting of approximately 10000 human genes to analyze differential gene expression at each time point. RESULTS: Forty-nine genes were found to be up-regulated and 37 genes were found to be down-regulated at various time points. Six genes were found to be consistently up-regulated at all time intervals, including collagen type 1 alpha-1 and alpha-2, 80K-L protein (MARCKS), and osteopontin. Six genes were found to be consistently down-regulated, including smoothelin and tropomyosin 2. RT-PCR and immunohistochemistry confirmed the microarray data. CONCLUSIONS: This study uses microarray analysis to identify genes that were temporally up- and down-regulated after prosthetic arterial grafting. Genes with similar patterns of expression have been identified, providing insights into related cellular pathways that may result in the formation of anastomotic intimal hyperplasia.

Temporal genomics of vein bypass grafting through oligonucleotide microarray analysis.

OBJECTIVE: Autologous vein is the conduit of choice for small artery reconstruction. Despite excellent patency, these conduits undergo remodeling over time. The purpose of this study was to identify temporal gene expression in vein grafts versus control veins through microarray analysis. METHOD: Cephalic vein grafts (n = 12) were used to bypass femoral arteries in canines. Vein grafts were harvested after 1, 7, 14, and 30 days. Normal contralateral cephalic vein served as control. Total RNA was isolated; its quantity and quality were confirmed with spectrophotometry and gel electrophoresis. Affymetrix U133A GeneChips, comprising approximately 15,000 genes, were used to analyze differential gene expression at each time point. Statistical analysis was performed with Affymetrix and dChip software to identify consistently upregulated and downregulated genes. Real-time, quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) and immunohistochemistry were used to validate microarray data. RESULTS: Statistical analysis revealed that 49 genes were consistently upregulated and 31 genes were consistently downregulated in all three animals at various time points. qRT-PCR to quantitatively assess messenger RNA expression was performed on specific genes to validate the microarray data. Immunohistochemistry to qualitatively assess protein expression was used for further validation. Hierarchical clustering with dChip identified additional genes with similar temporal or functional expression patterns. CONCLUSIONS: This is the first study to use microarray analysis with confirmatory qRT-PCR to identify altered genes after vein bypass grafting. Oligonucleotide microarrays and hierarchical clustering are powerful tools to generate hypotheses as the basis for additional research on gene expression in vein graft remodeling. Ultimately, identification of a temporal sequence of differential gene expression may provide insights not preferred into the molecular mechanisms of vein graft remodeling, but also into the pathways leading to intimal hyperplasia.