Mechanical forces are important regulators of cell function in many tissues including, for example, bone and components of the cardiovascular system. The endothelial lining of blood vessels has been shown to respond in an atheroprotective manner to unidirectional, laminar flow-induced shear stress and in an atherogenic manner to oscillating and low levels of shear. We have developed a cone and plate shear apparatus to simulate fluid shear stress on endothelial cells in vitro. The significant feature of this apparatus is that, unlike other in vitro flow systems, it accurately produces varying levels of shear stress, consistent with those created in vivo during the cardiac cycle. Flow characteristics of this system were verified by computational fluid dynamics (CFD) and laser Doppler velocimetry (LDV). Cellular responses were monitored by cell morphology and protein expression. These responses are consistent with in vivo responses as well as previous work using other in vitro flow systems.

Cardiovascular disease (CVD) is the leading cause of death in the United States. Analysis of vascular reactivity (VR) in response to brachial artery occlusion is used to estimate arterial health and to determine the likelihood of future cardiovascular complications. Development of a sensitive technique to assess VR is fundamental to the field of preventive cardiology. The conventional technique to study VR is by monitoring arterial diameter changes during hyperemia following occlusion using ultrasound based methods. Such measurements require highly qualified technicians and expensive equipment; and are complicated by signal noise introduced by motion and posture among others. It is well known that tissue temperature changes are a direct response to variations in blood flow, and it has been observed in small clinical studies that variations in fingertip temperature during brachial artery occlusion and subsequent hyperemia is a simple surrogate for the measurement of vascular reactivity and endothelial dysfunction. Given the promising nature of thermal monitoring to study VR, this thesis focuses on the analysis of the underlying physics affecting fingertip temperature during vascular occlusion and subsequent hyperemia. I will quantify the contribution of hemodynamic, anatomical and environmental factors over digit temperature changes, which will serve to determine the sensitivity of the digital thermal monitoring (DTM) technique. I have quantified the effect of several contributing factors to fingertip temperature and DTM results. The aims of this thesis focus on: (1) creation of a mathematical model of heat transfer at baseline, during, and after a reactive hyperemia test; and (2) validation of the model and experimental analysis of thermal and flow parameters in healthy volunteers. The proposed project is an innovative study that intends to show and quantify the relationship between VR and digital thermal reactivity, translating mathematical models based on the physics of heat transfer and fluid mechanics to clinical application. The parametric studies performed with the zeroth order model served to separate the contribution of environment and blood flow over the temperature curves measured during brachial artery occlusion. The thermal models developed were able to reproduce the trend of the temperature response observed experimentally at the fingertip.

Aging is associated impaired endothelial function in the skeletal muscle vasculature which contributes to decreased ability to increase muscle blow during exercise. This endothelial dysfunction is mediated, primarily, by impairments in the nitric oxide (NO) pathway in the skeletal muscle vasculature. The major purpose of this dissertation is to determine the mechanisms that mediate age-related endothelial dysfunction in rat soleus feed artery (SFA) and determine whether exercise training ameliorates this impairment in endothelial function. Therefore in these series of studies we sought to test three major hypotheses: 1) That exercise training reverses age-related decrements in endothelium-dependent dilation in SFA and that this improved endothelium-dependent dilation is the result of increased NO bioavailability due to increased content and phosphorylation of eNOS and/or increased antioxidant enzyme content; 2) That age-related endothelial dysfunction in rat SFA is mediated in part, by NAD(P)H oxidase-derived reactive oxygen species (ROS); 3) and, that impaired endothelium-dependent dilation in senescent SFA is due to an impaired potential for p-eNOSser1177. To test these hypotheses, SFA from young (4 month) and old (24 month) Fischer 344 rats were isolated for either determination of endothelium-dependent and –independent dilations or biochemical analyses. Results from these investigations suggest that 1) exercise training reverses the detrimental effects of aging on endothelial function in skeletal muscle feed arteries by enhancing the capacity to scavenge superoxide, increasing the bioavailability of NO; 2) ROS contribute to impaired endothelium-dependent dilation in old SFA; whereas, ROS appear to play a role in ACh-mediated dilation in SFA from young rats; 3) and, that the PI3 kinase/protein kinase B (Akt)/eNOS pathway is preserved with age.

名古屋大学博士学位論文 学位の種類 : 博士(医学)(論文) 学位授与年月日:平成5年9月14日 水谷大裕氏の博士論文として提出された

High-dose morphine has been extensively used in the control of postoperative and cancer pain. Patients receiving prolonged administration of high-dose morphine are known to be associated with certain cardiovascular complications and tissue regeneration defects. This research thesis aims to investigate the biological effects and molecular mechanisms of high-dose morphine on the vascular endothelial function, angiogenesis and wound regeneration using murine models of morphine-dependence and cultured endothelial cell assays. Mice were subjected to placebo or morphine (20 mg/kg, i.p.) injection for consecutive 14 days. Aortas were harvested for assessment of vasomotor function by isometric force recordings. Protein expression p47phox (a major subunit of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase) was determined by Western blotting. Generation of superoxide anions was detected under confocal microscope. Endothelium-dependent relaxations to acetylcholine were significantly reduced in morphine-treated animals, but were normalized by superoxide scavenging. Fluorescent densities of dihydroethidium and expression of p47phox were increased in the aorta of morphine-treated mice. In the second part of this thesis, the candidate determined the effects of high-dose morphine on angiogenesis and mobilization of endothelial progenitor cells (EPCs) in a mouse model of excisional wound injury. Excisional wound was created on control and morphine-dependent mice. Wound healing was compared by measuring the final-to-initial wound area ratio. Generation of superoxide anions in the wound was determined by luminol-enhanced chemiluminescence. Circulating mononuclear cells were isolated and measured for EPC (defined as CD34+/CD133+ cell) counts. In vivo and in vitro measurements of angiogenesis following morphine treatment were performed using the Matrigel assay. The results showed that wound closure was significantly reduced in mice treated with morphine when compared with controls, and higher levels of superoxide anions were generated in these wounds. High-dose morphine reduced numbers of circulating EPCs following creation of excisional wound. Matrigel assay showed impaired angiogenesis in animals and reduced capillary tube formation in cultured endothelial cells treated with high-concentration of morphine. Collectively, this research thesis demonstrated a number of novel findings. First, high-dose of morphine impairs vascular endothelial function by increased production of vascular superoxide anions. Activation of NADPH oxidase may be the molecular mechanisms responsible for reduced bioavailability of endothelium-derived NO. Second, systemic administration of high-dose morphine delays healing of excisional wounds and impairs angiogenesis. This antiangiogenic effect is associated with increased superoxide anions production and impaired mobilization of EPCs. In line with direct endothelial dysfunction, impaired angiogenesis and EPC mobilization resulted from high-dose morphine treatment may cause increased cardiovascular morbidity in human subjects receiving higher therapeutic dose of morphine.

ABSTRACT Endothelial dysfunction/apoptosis is critical to the development of neointima hyperplasia. Our pilot study identified a novel member of the caspase subfamily, caspase 13 (CASP 13), which is induced by oxidized low density lipoprotein (ox-LDL) in endothelial cells, but not in vascular smooth muscle cells. It remains controversial whether CASP 13 is a human or bovine gene. In this study, we collected four human brachial artery segments during arterioveinous fistula graft removal surgeries. By western blot and quantitative RT-PCR analysis, CASP13 expression was elevated at the anastomosis of brachial arteries from patients receiving hemodialysis. Rat carotid artery balloon model is widely used as in vivo atherosclerosis model for studies on neointima formation and especially vascular smooth muscle proliferation. However, most of the balloon injury studies terminated on the 14th day to sacrifice the animals for histological studies. In this study, we employed the ultrasonic device to record the hemodynamic changes in rat carotid artery at different time intervals after balloon injury. The pre-operative mean left carotid artery internal diameter and blood flow of Sprauge Dawley rats was 0.6 ¡Ó 0.07 mm and 28.75 ¡Ó 4.45 cm/sec, respectively. After balloon dilatation, the mean internal diameter of left carotid artery elevated to 0.77 ¡Ó 0.09 mm and 0.71 ¡Ó 0.08 mm on day 7 and day 14, respectively. Besides, the mean blood flow velocity also increased to 47.6 ¡Ó 9.2 cm/sec and 33.4 ¡Ó 10.8 cm/sec on day 7 and day 14, respectively. However, the left carotid artery blood flow velocity returned to 24 ¡Ó 3.5 cm/sec on day 21. The CASP 13 protein expression was found elevated in the balloon injury sites and mainly localized in the endothelial cells. In summary, CASP 13 is detected in endothelial cells in both human and rat atherosclerosis models and may constitute a novel molecular target for vascular diseases.

Endothelial Progenitor Cells (EPCs) were first identified from human blood samples as a population of circulating mononuclear cells capable of displaying a mature endothelial cell phenotype in culture. Subsequent studies have established that EPCs arise from the bone marrow (BM) and incorporate into the endothelium at sites of blood vessel growth, suggesting a potential role for these cells in neovascularization. Furthermore, a decline in EPC count has been correlated to multiple vascular pathologies, indicating that EPC number could serve as a biomarker of cardiovascular disease. Unfortunately, due to the variability in techniques used for EPC isolation and identification, considerable heterogeneity exists within the population of cells commonly defined as EPCs. In order for the clinical potential of EPCs to be fully realized, thorough characterization of the BM-derived cell populations involved in neovascularization is required. The objective of our study was to determine the functional significance of circulating EPCs in postnatal vascular growth and repair. Two separate strategies were employed to achieve this objective. In the first, we attempted to generate a novel mouse model where the pool of bone marrow-derived endothelial precursors was drastically reduced or eliminated. Our overall approach was to deliver a "suicide" gene, under control of an endothelial cell-specific promoter, to bone marrow cells for use in bone marrow transplantation (BMT) experiments. Mice receiving BMTs would therefore lack the ability to deliver viable BM-derived EPCs to sites of neovascularization. Our central hypothesis for this study was that a reduction in EPC viability would hinder endogenous vascular repair mechanisms, thereby exacerbating cardiovascular disease. In the second strategy, we attempted to identify novel progenitor cell populations based on the transcriptional regulation of pro-angiogenic genes. Our overall approach was to transduce BM with a retrovirus containing a fluorescent reporter gene under control of pro-angiogenic promoters for use in transplantation experiments. Our central hypothesis for this study was that unique populations of BM-derived cells could be identified by expression of the fluorescent reporter gene directed by the Vascular Endothelial Growth Factor (VEGF), endothelial Nitric Oxide Synthase (eNOS) and Vascular Endothelial (VE) Cadherin promoters. The BMT strategy utilized to address our first hypothesis was unsuccessful due to the use of a truncated form of the pro-apoptotic Bax as our suicide gene target. A plasmid encoding GFP fused to the truncated Bax fragment (ΔN-Bax, consisting of amino acids 112-192 of the full length protein) was used in transfection experiments to assess ΔN-Bax function. The GFP:ΔN-Bax fusion protein formed distinct extranuclear aggregates (presumably due to mitochondrial translocation) but did not induce apoptosis in transfected cells. The ΔN-Bax fragment also did not induce cell death when targeted to endothelial cells with retoviral-mediated gene delivery or in a transgenic mouse setting. To address our second hypothesis, we generated retroviral vectors containing the fluorescent tdTomato reporter under control of the VEGF, eNOS and VE Cadherin promoters. Significant fluorescence was detected in cultured endothelial cells and ex vivo-expanded BM cells. Following transplantation of transduced BM cells into lethally irradiated recipient mice, we were able to identify circulating populations of tdTomato-positive cells using flow cytometry. With these results we have identified novel subpopulations of circulating BM-derived cells which may play a significant role in post-natal neovascularization in mice. Therefore, results acquired from these studies could lead to improved cell therapy techniques for treatment of vascular disease.

This study explored the possibility that upregulation of Hypoxia Inducible Factor-1 (Hif-1)-responsive genes in Human Umbilical Vein Endothelial Cells (HUVEC) would promote and stabilize HUVEC formation into inchoate vascular beds within artificial collagen gels. This experiment was designed to explore the above possibility by sub-cloning Hif-1[alpha], the related chimeric construct Hif-1[alpha]/VP16, and the marker gene dsRed into retroviral expression vectors, producing retroviral vectors containing these genes, and stably transducing HUVEC using these retroviruses. Transduced HUVEC were to be observed in cell culture as well as after implantation into artificial collagen gels that have previously supported vascular bed formation by HUVEC. Our results show, preliminarily, that HUVEC transduced with Hif-1[alpha]/VP16 go into cell-cycle arrest. Attempts to transduce HUVEC with Hif-1[alpha] failed to achieve high enough transduction efficiency to determine the cells angiogenic potential. This study concluded that more experiments need to be conducted to better characterize the effects of hypoxia-responsive gene upregulation in controlling HUVEC angiogenesis and cell-cycle signaling and that straightforward transduction of HUVEC by Hif-1[alpha]/VP16 is probably not sufficient, in itself, to induce in vitro vascular bed formation.

Regeneration of the tissues to replace diseased, missing and traumatized dentin/pulp requires combining the recent progress in stem cell and tissue engineering research. Dental pulp stem cells (DPSCs) are considered as a promising population of cells in regenerative dentistry and shown to be able to produce dentin/pulp-like tissues following implantation in-vivo. Securing a good blood supply is critical in pulp regeneration, however, this is a challenging task due to the unique structure of the tooth, the anatomy of which permits only a microcirculatory system via a very small apical opening (<0.3-1mm). This limitation raises the need to develop novel methods to enhance angiogenesis during pulp regeneration. It was shown that DPSCs reside in the microvasculature region of the dental pulp and interact with perivascular cells. Therefore, endothelial cells could be a major source of modulators of pulp-dentin development and angiogenesis. If a pulp tissue substitute with pre-formed endothelial network could be engineered in-vitro, it would not only gain rapid anastomosis with host vasculature but also regulate DPSC function in pulp regeneration. In this study, for the first time, synergistic effects of DPSCs and human umbilical vein endothelial cells (HUVECs) on osteo/odontogenic differentiation and angiogenesis were investigated using two-dimensional and three-dimensional direct co-culture systems. Furthermore, the potential of three-dimensional DPSC constructs prevascularized with HUVECs in dental pulp regeneration in-vivo was exmined. HUVECs promoted odonto/osteogenic differentiation of DPSCs in direct two-dimensional co-cultures in-vitro. Further, addition of DPSCs stabilized the pre-existing vessel-like structures formed by HUVECs and increased the longevity of these structures on matrigel in-vitro. Using two different systems, scaffold-free self-assembling microtissue spheroids and peptide hydrogel scaffold, the interactions of DPSCs and HUVECs in three-dimensional cultures were investigated. The results demonstrated that DPSCs can self assemble into three-dimensional microtissue spheroids when cultured alone or with HUVECs. DPSCs promoted survival and vascular structure formation by HUVECs both in scaffold-free microtissue spheroids and peptide hydrogel scaffold. In contrast, HUVECs, when cultured alone, neither formed vascular structures nor survived in either of the 3D systems. The latter phenomenon was attributable to vascular endothelial growth factor secreted by DPSCs, a major factor responsible for endothelial function. Co-cultures also showed enhanced odonto/osteogenic differentiation in both three-dimensional microtissue spheroid and peptide hydrogel scaffold systems. Following implantation of tooth-root fragments filled with three-dimensional DPSC constructs into the subcutaneous space of immunodefficient mice, vascularised pulp-like tissue was regenerated within the root canals. Compared to DPSC-only group, DPSC/HUVEC co-culture groups showed higher vascularisation, extracellular matrix formation and mineralization in regenerated tissue. More importantly, HUVEC-lined vascular lumens were observed in regenerated tissues suggesting the successful integration of in-vitro formed pre-vascular structures to the host vasculature. In summary, the findings suggest that DPSCs and HUVECs display significant synergy during odonto/osteogenic differentiation and angiogenesis when co-cultured either in two-dimensional or three-dimensional culture systems. Unravelling these fundamental behavioural patterns of DPSCs provides novel insights into the process of pulp regeneration, leading to new avenues for more effective therapies in regenerative endodontics. / published_or_final_version / Dentistry / Doctoral / Doctor of Philosophy

Uncontrolled cell growth may lead to pathological conditions such as cancer. During the progression of cancer, cancer cells stimulate endothelial cells for angiogenesis to support their growth and migration. Previous studies suggest that Astragalus membranaceus, of which the dried root [Astragali Radix] is used as a traditional Chinese medicine, and its bioactive components, astragalus saponins (AST), astragaloside IV (AS IV) and isoflavonoid calycosin, inhibit cancer growth. The present study aimed to examine whether or not these components inhibit the growth and/or metastasis of colon cancer cells and/or angiogenesis of endothelial cells, and to determine the possible mechanisms involved. The growth of HCT 116 colon cancer cells and human umbilical vein endothelial cells (HUVEC) after 72 hours incubation with AST (1 to 25 μg/ml), AS IV (0.5 to 100 μM) or calycosin (10 to 200 μM) were detected with thiazolyl blue tetrazolium bromide assay. Wound healing migration and tube formation assays were used to examine the metastatic and angiogenic potential of HCT 116 cells and HUVEC. Moreover, the expressions of apoptotic [B-cell lymphoma 2 and procaspase-3] and metastasis/angiogenesis-related proteins [matrix metalloproteinase (MMP)-2, MMP-9 and vascular endothelial growth factor (VEGF)] were measured with Western immunoblotting. To investigate the potential mechanism(s) through which astragalus components affect the proliferation and/or migration of HCT 116 cells and HUVEC, the activities of mitogen-activated protein (MAP) kinases [extracellular signal-regulated kinase 1 and 2 (ERK1/2), p38 MAP kinase (p38) and c-Jun amino-terminal kinases] were studied by measuring the expressions of their phosphorylated and total proteins with Western immunoblotting. Calycosin (200 μM) inhibited the growth of HCT 116 cells without affecting that of HUVEC. While it inhibited the migration of both cell types, it stimulated tube formation only in HUVEC. In HCT 116 cells, calycosin downregulated the expressions of procaspase-3, VEGF, MMP-2 and MMP-9 proteins, inhibited ERK1/2 but activated p38. These effects of calycosin were not observed in HUVEC. Neither AST nor AS IV had any significant effects on the parameters studied in HCT 116 cells. AST also showed no effect in HUVEC; AS IV, at 100 μM, appeared to increase the number of tube formation by HUVEC. In conclusion, the present findings suggest that AST has no significant effect on both cancer and endothelial cells while AS IV may promote angiogenesis without any direct action in colon cancer cells. In colon cancer cells, calycosin induces apoptosis, possibly through activation of caspase-3 and p38, and inhibits metastasis, possibly by downregulating MMP-2 and MMP-9, and inhibiting ERK1/2. However, in endothelial cells, the effect of calycosin is not conclusive as it promotes tube formation but inhibits migration. These findings provide the pharmacological basis for the use of Astragali Radix in the treatment of colon cancer, and the scientific evidence for a therapeutic potential of calycosin in the management of this disorder. Further studies are needed to verify the effect of calycosin on endothelial cells. In order to better mimic the clinical situation, the interaction between cancer and endothelial cells [for example, tumor-induced angiogenesis] needs to be taken into consideration. / published_or_final_version / Pharmacology and Pharmacy / Master / Master of Philosophy