Blood vessels mimics (BVMs) are tissue-engineered blood vessels used to test vascular devices in an environment that mimics some simple anatomical factors of native blood vessels. It is important to accurately and consistently assess tissue-engineered blood vessels, although there is currently a lack of standardization in Cal Poly’s Tissue Engineering Lab and in the entirety of the field. The goal of this thesis was to develop and optimize imaging and image quantification techniques for tissue-engineered blood vessels. The first aim of this thesis optimized and compared imaging and assessment techniques for electrospun scaffolds. Images from different SEMs were compared to determine the benefits and drawbacks of each microscope. Several materials were also imaged using these microscopes to characterize polymers at the microscopic scale and to compare the quality of images from different SEMs. The second aim of this thesis validated and implemented a MATLAB-based automatic fiber diameter measurement tool. Fiber measurements were obtained from a manual ImageJ method, a semi-automatic DiameterJ method, and a new automatic MATLAB method and compared to evaluate accuracy and user variability of the MATLAB tool. The results of this aim validated the accuracy of the MATLAB tool and showed that it resulted in lower user variability as compared to other fiber diameter measurement methods. The third aim of this thesis developed imaging techniques for novel silicone BVMs at each stage of development. Evaluation techniques to quantify cell adhesion and coverage on silicone BVMs using SEM, widefield fluorescent imaging, and immunochemistry were developed. After refining those methods, they were applied and adapted to silicone BVMs with deployed devices. BBI, H&E, and PECAM-1 staining were all found to be effective assessment methods for silicone BVMs. Overall, the work described in this thesis increased the consistency, standardization, and accuracy of scaffold and BVM assessment in Cal Poly’s Tissue Engineering Lab.

La neuroinflammation est un dénominateur commun de plusieurs troubles du système nerveux central. Les réactions inflammatoires sont souvent médiées par plusieurs voies de signalisation qui conduisent à l'ouverture de la barrière hémato-encéphalique. L'activateur tissulaire du plasminogène (tPA) est une serine protéase qui induit l'ouverture de la barrière hémato-encéphalique. Au cours des dernières années, il a également été montré que les récepteurs NMDA situés dans les cellules endothéliales peuvent jouer un rôle crucial dans la propagation de la réaction inflammatoire.Mon travail au cours de ma thèse a mis l'accent sur la découverte des mécanismes par lesquels le récepteur NMDA effectue une médiation de l'ouverture de la barrière hémato-encéphalique induite par le TPA. Dans notre première étude, nous montrons que les récepteurs NMDA endothéliaux sont des cibles thérapeutiques potentielles pour prévenir l'infiltration et l'inflammation des cellules immunitaires médiées par l'EAE. Nous montrons que l'anticorps monoclonal du récepteur NMDA spécifique à la souris, le Glunomab, pourrait protéger la barrière de la moelle épinière de dommages inflammatoires. Nous montrons également que les récepteurs NMDA sont exprimés en étroite association avec les protéines de jonction serrées dans les cellules endothéliales cérébrales. Dans notre deuxième étude, nous montrons pour la première fois que les récepteurs NMDA neuroendothéliaux peuvent présenter une action métabotropique lors de l'inflammation. Nous soulignons également que ces récepteurs sont en effet des récepteurs NMDA non conventionnels exprimant la sous unité GluN3A. En outre, nous rapportons que le tPA accélère l'ouverture de la barrière hémato-encéphalique en présence d'une agoniste rare de la glycine par un mécanisme dépendant de l'activation de RhoA. Les résultats de mon projet apportent une nouvelle vision du rôle des récepteurs NMDA métabotropiques dans les cellules endothéliales cérébrales. En outre, il fournit également des détails plus précis sur l'ouverture de la barrière hémato-encéphalique via l’activateur tissulaire du plasminogène. / Neuroinflammation is a common denominator of several central nervous system disorders. Inflammatory reactions are often mediated by several signaling pathways which lead to the opening of the blood brain barrier. Tissue plasminogen activator (tPA) is a serine protease induces opening of the blood brain barrier. In recent years, it has also been shown that NMDA receptors located in endothelial cells can play a crucial role in propagation of inflammatory reaction. My doctoral study focused on the finding the underlying mechanisms of action(s) by which NMDA receptor mediates tPA induced opening of the blood brain barrier. In our first study we show that endothelial NMDA receptors are potential therapeutic targets to prevent EAE mediated immune cell infiltration and inflammation. We show that NMDA receptor specific mouse monoclonal antibody Glunomab could prevent the brain spinal cord barrier from inflammatory damage. We also show that NMDA receptors are expressed in close association of tight junction proteins in cerebral endothelial cells. In our second study, we show for the first time that, neuroendothelial NMDA receptors can exhibit metabotropic mode of action during inflammation. We also highlight that these receptors are indeed GluN3A expressing non-conventional NMDA receptors. In addition, we report that tPA accelerates the opening of blood brain barrier in presence of an uncommon agonist glycine by RhoA activation dependent mechanism.My project results provide a nouvelle insight for the role of metabotropic NMDA receptors in cerebral endothelial cells. In addition it also provides more precise details of blood brain barrier opening mediated by tissue plasminogen activator.

Cardiovascular disease is currently the leading cause of death in the U.S. that frequently requires bypass surgery using vascular grafts for treatment. Current limitations with fully synthetic grafts have led researchers to bioengineered alternatives that consist of a combination of vascular scaffolds and cells. A major challenge in creating a functional bioengineered vascular graft is development of a confluent endothelium on the lumen that is able to resist detachment under physiologic fluid flow. In addition, methodologies used to assess the growth and maturation of the endothelium in a noninvasive and dynamic manner are severely lacking. Therefore, the overall goal of this research is to advance the field of vascular tissue engineering by 1) creating methodologies to enhance EC adherence to a vascular graft and 2) development of a noninvasive and real-time imaging system capable of assessing the graft endothelium.  To achieve these objectives, three separate studies were performed. In the first study, electrospun scaffold fiber diameter and alignment were systematically varied to determine their effect on endothelial cell (EC) morphology and adherence under fluid flow. ECs on uniaxially aligned nanofibers displayed elongated and aligned morphologies leading to higher adherence to the scaffolds under physiologic levels of fluid flow as compared to those on randomly oriented scaffolds. In the second study, a fiber optic based (FOB) imaging system was developed to image fluorescent ECs through a thick electrospun scaffold.  Results demonstrated that the FOB imaging system was able to accurately visualize fluorescent ECs in a noninvasive manner through the thick and highly opaque scaffold. In the final study, the FOB imaging system was used to noninvasively quantify vascular graft endothelialization, EC detachment, and apoptosis through the vessel wall with greater imaging penetration depth than two-photon microscopy. Additionally, the FOB method was capable of continuously tracking EC migration and endothelialization of a bioengineered graft in a bioreactor. Overall, these results demonstrate that aligned scaffold topographies enhance EC adherence under fluid flow and the FOB imaging system is a promising tool to monitor endothelium development and response to fluid flow in a manner that has not previously been afforded using conventional imaging methods. / Ph. D.

Endothelial Progenitor Cells (EPCs) are thought to participate in the healing of injured vascular endothelium by incorporating into the defect sites to mediate endothelial recovery. Recently, osteopontin (OPN) was shown to be fundamental in accelerating estrogen-dependent healing of injured blood vessels. Here, we are investigating the effect OPN has on EPC behavior. Late outgrowth human EPCs (LEPCs) were derived from circulating monocytes isolated by leukophoresis, and grown in culture until passage six. L-EPCs were then assayed for adhesion, spreading, chemotaxis, and haptotaxis, as well as resistance to detachment by flow electric cellsubstrate impedance sensing (ECIS). The results of standard and ECIS methods showed both dose and time dependent responses in cell adhesion and spreading. In addition, OPN promoted haptotactic migration of EPCs in Boyden chamber assays. LEPCs seeded onto 10μM OPN substrates and exposed to laminar flow had grater survival and higher resistance to detachment than OPN/static and flow only conditions. CD44 and !1 integrins were only responsible for approximately 50% of LEPCs adhesion to OPN compared to the unblocked condition. Western blots showed that Rho GTPases were activated in L-EPCs seeded on OPN. However, this activation could not be completely blocked by either CD44 or !1 integrin antagonists. These data confirm the direct effects of OPN on EPCs adhesion, and suggest that OPN works by mediating cell adhesion during vascular injury.

Objective: We proposed that women who develop preeclampsia have a low ratio of 'protective' toxicity preventing activity (TxPA) to 'toxic' very low density lipoproteins (VLDL) late in pregnancy. Having confirmed this hypothesis, we then tested whether this low ratio would manifest itself early in the pregnancy of women who develop preeclampsia. Methods: Serially collected plasma from women who developed preeclampsia and from matched controls was assayed blind for TxPA, triglycerides, cholesterol, high-density lipoproteins, albumin, and nonesterified fatty acids (NEFA). Main Outcome Measures: Plasma concentrations of lipids, NEFA, and proteins which bind NEFA (TxPA and albumin) were measured in normal and preeclamptic women. These parameters were formulated prior to data collection because of the low albumin/triglyceride' ratios and the elevated NEFA levels reported to occur in preeclampsia. Results: In late pregnancy, TxPA was lower (1.82 ± 0.63 vs. 2.30 ± 0.40 g/dL, P = 0.008) and VLDL higher (292 ± 130 vs. 206 ± 60 mg/dL, P = 0.013) in preeclamptics than in controls. Discrimination analysis (TxPA and triglyceride), correctly classified 95% of the preeclamptics and 79% of the controls in late pregnancy. The ratio of TxPA to non-TxPA and triglyceride correctly classified 92% of the preeclamptics and 85% of the controls in early pregnancy. Conclusions: The ratio of TxPA to VLDL accurately distinguishes preeclamptic from normal pregnant women, suggesting that both these factors are involved in the development of preeclampsia.

A zebrafish transcript dubbed GA2692 was initially identified via a whole-mount in situ hybridization screen for vessel specific transcripts. Its mRNA expression during embryonic development was detected in ventral hematopoietic and vasculogenic mesoderm and later throughout the vasculature up to 48 hours post fertilization. Morpholino-mediated knockdown of GA2692 in embryos resulted in multiple defects in vasculature, particularly, at sites undergoing active capillary sprouting: the intersegmental vessels, sub-intestinal vessels and the capillary sprouts of the pectoral fin vessel. During the course of these studies, a homology search indicated that GA2692 is the zebrafish orthologue of mammalian HspA12B, a distant member of the heat shock protein 70 (Hsp70) family. By a combination of northern blot and realtime PCR analysis, we showed that HspA12B is highly expressed in human endothelial cells in vitro. Knockdown of HspA12B by small interfering RNAs (siRNAs) in human umbilical vein endothelial cells blocked wound healing, migration and tube formation, whereas overexpression of HspA12B enhanced migration and accelerated wound healing - data that are consistent with the in vivo fish phenotype obtained in the morpholino-knockdown studies. Phosphorylation of Akt was consistently reduced by siRNAs against HspA12B. Overexpression of a constitutively active form of Akt rescued the inhibitory effects of knockdown of HspA12B on migration of human umbilical vein endothelial cells. Collectively, our data suggests that HspA12B is a highly endothelial-cell-specific distant member of the Hsp70 family and plays a significant role in endothelial cells during development and angiogenesis in vitro, partially attributable to modulation of Akt phosphorylation.

OBJECTIVE - HSPA12B is the newest member of HSP70 family of proteins and is enriched in atherosclerotic lesions. This study focused on HSPA12B expression in mice and its involvement in angiogenesis. METHODS AND RESULTS - The expression of HSPA12B in mice and cultured cells was studied by: (1) Northern blot; (2) in situ hybridization; (3) immunostaining with HSPA12B-specific antibodies; and (4) expressing Enhanced-Green-Fluorescent-Protein under the control of the HSPA12B promoter in mice. The function of HSPA12B was probed by an in vitro angiogenesis assay (Matrigel) and a migration assay. Interacting proteins were identified through a yeast two-hybrid screening. HSPA12B is predominantly expressed in vascular endothelium and induced during angiogenesis. In vitro angiogenesis and migration are inhibited in human umbilical vein endothelial cells in the presence of HSPA12B-neutralizing antibodies. HSPA12B interacts with multiple proteins in yeast 2-hybrid system. CONCLUSIONS - We provide the first evidence to our knowledge that the HSPA12B is predominantly expressed in endothelial cells, required for angiogenesis, and interacts with known angiogenesis regulators. We postulate that HSPA12B provides a new mode of angiogenesis regulation and a novel therapeutic target for angiogenesis-related diseases.

The relationship between hyperlipidemia and sensorineural hearing loss remains obscure. In this study, we elucidate for the first time the cochlear morphological and auditory alterations and their relationships with hyperlipidemia, atherosclerosis, and endothelial dysfunction in apolipoprotein-E knockout (ApoE-KO) mice. Ten-week-old ApoE-KO mice were fed either atherosclerotic diet (1.25% cholesterol) or normal diet. Wild type mice (C57BL/6J) served as normal controls. Fourteen weeks later, marked hyperlipidemia, atherosclerosis, endothelial dysfunction, and hearing impairment, especially in the high frequencies, had developed in ApoE-KO mice as compared with C57BL/6J mice (P < 0.001). A high positive correlation between hearing loss and the extent of atherosclerosis and plasma total cholesterol levels was found. Hearing loss, especially at high frequencies, was detected in all ApoE-KO mice. Hair cell loss mainly at the base turn, thickening of vascular intima, and lumen stenosis of the spiral modiolar artery (SMA) in cochlea were also found; these histological changes were exacerbated by the atherosclerotic diet. Furthermore, endothelial nitric oxide synthase (eNOS) in aortic wall and cochlea was distinctly reduced in ApoE-KO mice. These results demonstrate that hyperlipidemia and atherosclerosis can induce alterations in cochlear morphology and function. The stenosis of SMA, which may cause cochlear ischemia and hypoxia, endothelial dysfunction, and low eNOS activity, may contribute to hearing loss.

Ameliorating cardiac microvascular injury is the most effective means to mitigate diabetes-induced cardiovascular complications. Inositol-requiring 1α (IRE1α), a sensor of endoplasmic reticulum stress, is activated by Toll like receptors (TLRs), and then promotes cardiac microvascular injury. Peli1 is a master regulator of TLRs and activates IRE1α. This study aims to investigate whether Peli1 in endothelial cells promotes diabetes-induced cardiac microvascular injury through activating IRE1α. Here we found that Peli1 was markedly up-regulated in cardiac endothelial cells of both diabetic mice and in AGEs-treated cardiac microvascular endothelial cells (CMECs). Peli1 deficiency in endothelial cells significantly alleviated diabetes-induced cardiac microvascular permeability, promoted microvascular regeneration, and suppressed apoptosis, accompanied by the attenuation of adverse cardiac remodeling. Furthermore, Peli1 deletion in CMECs ameliorated AGEs-induced damages in vitro. We identified heat shock protein 90 (Hsp90) as a potential binding partner for Peli1, and the Ring domain of Peli1 directly bound with Hsp90 to enhance IRE1α phosphorylation. Our study suggests that blocking Peli1 in endothelial cells may protect against diabetes-induced cardiac microvascular injury by restraining ER stress.

Etv2 is an endothelial-specific ETS transcription factor that is essential for endothelial differentiation and vascular morphogenesis in vertebrates. However, etv2 expression dynamics during development and the mechanisms regulating it are poorly understood. I found that etv2 transcript and protein expression are highly transient during zebrafish vascular development, with both expressed early during development and then subsequently downregulated. Inducible knockdown of Etv2 in zebrafish embryos prior to mid-somitogenesis, but not later, causes severe vascular defects, suggesting a role for Etv2 in specifying angioblasts from the lateral mesoderm. I further demonstrate that the 3’UTR of etv2 is post-transcriptionally regulated in part by the let-7 family of microRNAs. Ectopic expression of let-7a represses endogenous Etv2 transcript and protein expression with a concomitant reduction in endothelial cell gene expression. Additionally, overexpressed Etv2 in HEK293T cells is ubiquitinated and degraded by the proteasome. Accordingly, endogenous zebrafish Etv2 protein is rapidly degraded in the presence of the translation inhibitor cycloheximide in vivo. Taken together, our results suggest that etv2 acts during early development to specify endothelial lineages and is subsequently downregulated through post-transcriptional and post-translational mechanisms, to allow normal vascular development to proceed.