Browsing by Author "Truskey, George A"

Clinical studies have identified strong correlations between aging and the development of atherosclerosis. In particular, endothelial cell senescence is implicated in age-related changes in vasoreactivity. Oxidative stress is considered the primary source of endothelial cell (EC) senescence in vivo. EC senescence leads to abnormal proliferation of vascular smooth muscle cells, reduced vasoreactivity, enhanced vascular permeability, and greater adhesion of circulating monocytes and lipids. Endothelial senescence often occurs coincident with an inflammatory response within the endothelium. Recapitulating this mechanism of inducing EC senescence in vitro will facilitate a more precise understanding of how aging contributes to endothelial dysfunction and development of vascular diseases, particularly atherosclerosis. Additionally, evidence of vascular remodeling, particularly deposition of fibronectin and stiffening of the vessel wall matrix, is found in both older patients and atheroprone regions. The independent effects of these factors on the function of endothelial cells is poorly understood due to the inability to study them in isolation in vivo. The Truskey lab developed tissue-engineered blood vessels (TEBVs) which recapitulate the structure and function of an arteriole in vitro. These vessels can be fabricated rapidly, perfused immediately after fabrication, and reach functional maturity after a week. Measurements of endothelium-mediated vascular function confirm the presence of a healthy endothelium in the vessels for several weeks after initial fabrication. This in vitro system allows more precise control over the cellular and structural components of blood vessels than is possible with in vivo experiments. Ultimately, the development of a more robust in vitro model for atherosclerosis will contribute to an increased understanding of vascular disease progression and provide a platform for the evaluation of new drugs during preclinical trials. Specific Aim 1: Evaluate the functional effects of stress-induced senescence on TEBVs. Stress-induced senescence reduced endothelium-dependent vessel function and resulted in endothelial cell inflammation with minimal effects on the surrounding hNDFs. Stress-induced senescence was induced in vitro by treatment with hydrogen peroxide. 2-D cells and TEBVs were treated for 5 or 7 days with hydrogen peroxide. Cells in 2-D were stained for p21 to evaluate senescence, as well as key immune cell adhesion markers VCAM-1, ICAM-1, and E-Selectin. To characterize the effects on TEBVs, vasoreactivity in response to an endothelium-independent vasodilator (sodium nitroprusside) and vasoconstrictor (phenylephrine) were quantified, as well as endothelium-dependent vasoreactivity (acetylcholine). Immunostaining of p21 and VCAM-1 expression was also used to confirm that senescence and inflammation were induced in the TEBVs alongside the reduction in endothelium-dependent vasodilation. Specific Aim 2: Evaluate the capacity of stress-induced senescence to increase the monocyte adhesion and foam cell formation in the TEBVs Stress-induced senescence in TEBVs increased adhesion of circulating monocytes and foam cell formation in accumulated monocytes and medial hNDFs. Senescence was induced as in Aim 1, by treating vessels with hydrogen peroxide. The resulting increase in senescence, VCAM-1, and E-selectin increased adhesion of circulating monocytes to the vessel wall. To develop an atherogenesis model, low density lipoprotein was enzymatically modified into a more inflammatory state which is often identified within atherosclerotic lesions. Introducing enzyme-modified low-density lipoprotein (eLDL) alongside hydrogen peroxide treatment further increased endothelial cell activation. There was a significant increase in the percentage of ICAM-1 positive cells when eLDL was applied to endothelial cells alongside H2O2. hNDFs absorbed and retained eLDL, even without H2O2 in the growth media. When TEBVs were exposed to a combination of eLDL, H2O2, and cell-tracker red monocytes, endothelium-dependent vasoreactivity was significantly compromised. Lipid retention within the vessel wall was significant, as was adhesion of monocytes. Specific Aim 3: Evaluate the drug-responsiveness of the TEBV senescence model and the ability of geroprotective agents to reduce senescence-induced vascular dysfunction, monocyte adhesion, and foam cell formation. Development of a physiologically relevant model for vascular senescence can provide a valuable tool for evaluating the efficacy of drugs targeting atherosclerosis, particularly a new class of drugs in development called senolytics. Senolytics, and their sister drugs senomorphics, specifically target senescent cells and transiently disable the anti-apoptotic pathways that prolong their lives, reducing the burden of senescent cells within the tissue. Senomorphics target factors within the senescence-associated secretory pathway (SASP) to reduce cytokine production and inflammation. Dasatinib and quercetin, two senolytics, and tacrolimus, a senomorphic, were tested on CBECFCs growing in 2-D to see if they were effective at reducing the percentage of p21 positive (senescent) cells. Tacrolimus was found to be the best candidate and used in TEBV trials. TEBVs treated with tacrolimus for 48 hours after induction of senescence recovered significantly more endothelium-dependent vasoreactivity compared to vessels left to recover from H2O2 in normal growth media. Additionally, addition of tacrolimus for the duration of hydrogen peroxide treatment had an atheroprotective effect. Adhesion of monocytes and foam cell formation were significantly reduced compared to vessels without tacrolimus. In summary, the work presented here demonstrates that a TEBV model of vascular senescence can be generated in under two weeks using near-physiological levels of hydrogen peroxide. This model can be capitalized upon to model atherogenesis by adding only eLDL and monocytes. We were also able to effectively use the senomorphic tacrolimus to mitigate the effects of senescence on monocyte adhesion and lipid uptake. This system could be used to investigate other senolytics or test the efficacy and toxicity of novel drugs still in development.

The ability of tissue engineered constructs to replace diseased or damaged organs is limited without the incorporation of a functional vascular system. To design microvasculature that recapitulates the vascular niche functions for each tissue in the body, we investigated the following hypotheses: (1) cocultures of human umbilical cord blood-derived endothelial progenitor cells (hCB-EPCs) with mural cells can produce the microenvironmental cues necessary to support physiological microvessel formation in vitro; (2) poly(ethylene glycol) (PEG) hydrogel systems can support 3D microvessel formation by hCB-EPCs in coculture with mural cells; (3) mesenchymal cells, derived from either umbilical cord blood (MPCs) or bone marrow (MSCs), can serve as mural cells upon coculture with hCB-EPCs. Coculture ratios between 0.2 (16,000 cells/cm2) and 0.6 (48,000 cells/cm2) of hCB-EPCs plated upon 3.3 µg/ml of fibronectin-coated tissue culture plastic with (80,000 cells/cm2) of human aortic smooth muscle cells (SMCs), results in robust microvessel structures observable for several weeks in vitro. Endothelial basal media (EBM-2, Lonza) with 9% v/v fetal bovine serum (FBS) could support viability of both hCB-EPCs and SMCs. Coculture spatial arrangement of hCB-EPCs and SMCs significantly affected network formation with mixed systems showing greater connectivity and increased solution levels of angiogenic cytokines than lamellar systems. We extended this model into a 3D system by encapsulation of a 1 to 1 ratio of hCB-EPC and SMCs (30,000 cells/µl) within hydrogels of PEG-conjugated RGDS adhesive peptide (3.5 mM) and PEG-conjugated protease sensitive peptide (6 mM). Robust hCB-EPC microvessels formed within the gel with invasion up to 150 µm depths and parameters of total tubule length (12 mm/mm2), branch points (127/mm2), and average tubule thickness (27 µm). 3D hCB-EPC microvessels showed quiescence of hCB-EPCs (<1% proliferating cells), lumen formation, expression of EC proteins connexin 32 and VE-cadherin, eNOS, basement membrane formation by collagen IV and laminin, and perivascular investment of PDGFR-β+/α-SMA+ cells. MPCs present in <15% of isolations displayed >98% expression for mural markers PDGFR-β, α-SMA, NG2 and supported hCB-EPC by day 14 of coculture with total tubule lengths near 12 mm/mm2. hCB-EPCs cocultured with MSCs underwent cell loss by day 10 with a 4-fold reduction in CD31/PECAM+ cells, in comparison to controls of hCB-EPCs in SMC coculture. Changing the coculture media to endothelial growth media (EBM-2 + 2% v/v FBS + EGM-2 supplement containing VEGF, FGF-2, EGF, hydrocortisone, IGF-1, ascorbic acid, and heparin), promoted stable hCB-EPC network formation in MSC cocultures over 2 weeks in vitro, with total segment length per image area of 9 mm/mm2. Taken together, these findings demonstrate a tissue engineered system that can be utilized to evaluate vascular progenitor cells for angiogenic therapies.

Hutchison-Gilford Progeria Syndrome (HGPS) is a rare, accelerated aging disorder caused by nuclear accumulation of progerin, an altered form of the Lamin A gene. The primary causes of death are stroke and cardiovascular disease at an average age of 14 years. It is known that loss or malfunction of smooth muscle cells (SMCs) in the vasculature leads to cardiovascular defects, however, the exact mechanisms are still not understood. The contribution of other vascular cell types, such as endothelial cells, is still not known due to the current limitations of studying such a rare disorder. Due to limitations of 2D cell culture, mouse models, and the limited HGPS patient pool, there is a need to develop improved models of HGPS to better understand the development of the disease and discover novel therapeutics.

To address these limitations, we produced a functional, three-dimensional tissue model of HGPS that replicates an arteriole-scale tissue engineered blood vessel (TEBV) using induced pluripotent stem cell (iPSC)-derived cell sources from HGPS patients. To isolate the specific effects of HGPS SMCs, we initially used human cord blood-derived endothelial progenitor cells (hCB-EPCs) from a separate, healthy donor and iPSC-derived SMCs (iSMCs). TEBVs fabricated from HGPS patient iSMCs and hCB-EPCs (HGPS iSMC TEBVs) showed disease attributes such as reduced vasoactivity, increased medial wall thickness, increased calcification, excessive extracellular matrix protein deposition, and cell apoptosis relative to TEBVs fabricated from primary mesenchymal stem cells (MSCs) and hCB-EPCs or normal patient iSMCs with hCB-EPCs. Treatment of HGPS iSMC TEBVs for one week with the rapamycin analog Everolimus (RAD001), increased HGPS iSMC TEBV vasoactivity and iSMC differentiation in TEBVs.

To improve the sensitivity of our HGPS TEBV model and study the effects of endothelial cells on the HGPS cardiovascular phenotype, we adopted a modified differentiation protocol to produce iPSC-derived vascular smooth muscle cells (viSMCs) and endothelial cells (viECs) from normal and Progeria patient iPSC lines to create iPSC-derived vascular TEBVs (viTEBVs). Normal viSMCs and viECs showed structural and functional characteristics of vascular SMCs and ECs in 2D culture, while HGPS viSMCs and viECs showed various disease characteristics and reduced function compared to healthy controls. Normal viTEBVs had comparable structure and vasoactivity to MSC TEBVs, while HGPS viTEBVs showed reduced vasoactivity, increased vessel wall thickness, calcification, apoptosis and excess ECM deposition. In addition, HGPS viTEBVs showed markers of cardiovascular disease associated with the endothelium such as decreased response to acetylcholine, increased inflammation, and altered expression of flow-associated genes.

The treatment of viTEBVs with multiple Progeria therapeutics was evaluated to determine the potential of the HGPS viTEBV model to serve as a platform for drug efficacy and toxicity testing as well as to further elucidate the mechanisms behind each drugs mode of action. Treatment of viTEBVs with therapeutic levels of the farnesyl-transferase inhibitor (FTI), Lonafarnib, or Everolimus improved different aspects of HGPS viTEBV structure and function. Treatment with Everolimus alone increased response to phenylephrine, improved SMC differentiation and cleared progerin through autophagy. Lonafarnib improved acetylcholine response, decreased ECM deposition, decreased calcification and improved nitric oxide production. Most significantly, combined therapeutic treatment with both drugs showed an additive effect by improving overall vasoactivity, increasing cell density, increasing viSMC and viEC differentiation, and decreasing calcification and apoptosis in treated HGPS viTEBVs. On the other hand, toxic doses of both drugs combined resulted in significantly diminished HGPS viTEBV function through increased cell death. In summary, this work shows the ability of a tissue engineered vascular model to serve as an in vitro personalized medicine platform to study HGPS and potentially other rare diseases of the vasculature using iPSC-derived cell sources. It has also further identified a potential role of the endothelium in HGPS. Finally, this HGPS viTEBV model has proven effective as a drug testing platform to determine therapeutic and toxic doses of proposed therapeutics based on their specific therapeutic effects on HGPS viTEBV structure and function.

The purpose of this work is to determine if branched chain amino acids (BCAA) could have a causative role in the development of atherosclerosis. Atherosclerotic lesions occur in the vasculature and mediate the progression of cardiovascular disease (CVD). Advanced atherosclerotic lesions can lead to heart attacks or strokes. Conflicting evidence from previous studies has made it difficult to understand if BCAA help or hurt cardiovascular health, and it is not known if other pro-atherosclerotic factors cooperate with BCAA to accelerate atherosclerosis. While studies in human patients have shown that there is a correlation between BCAA levels in the blood and the development of diseases like metabolic syndrome and CVD, we cannot conclude that BCAA cause these diseases from association studies alone. Additionally, some studies in animals have shown that supplementation with BCAA supports cardiovascular health. Therefore, we need to determine if there is a mechanistic link between BCAA levels and atherosclerotic disease processes in human cells. This will also help us determine if BCAA could be a mechanistic link between metabolic syndrome and CVD.

In this work, we use a tissue engineered blood vessel (TEBV) model to determine the role of BCAA in the development of atherosclerosis. The TEBV model is an artificial blood vessel made of a collagen-based scaffold and populated with vascular cells, using similar tissue architecture and environmental stimuli of an artery in the human body. The TEBV system models the processes that occur in atherosclerosis, such as inflammation, loss of vasomotor tone, and interactions between white blood cells and vascular cells. Measuring these processes then allows us to predict what effect a novel risk factor, such as BCAA, would have on vascular health in the human body.

In chapter 2, we develop an “intermediate stage” lesion model in the TEBV system. Atherosclerotic lesions develop over many years, and since metabolic syndrome is often diagnosed in adults, many patients will have existing lesions. Therefore, it is important to expand upon existing models of early atherogenesis to include features that occur in later disease stages, such as remodeling of the vessel wall. We recapitulate the increase of a carbohydrate-based molecule called chondroitin sulfate (CS) that occurs in the atherosclerotic extracellular matrix in our TEBV model. While many studies have shown that CS enhances the development of atherosclerosis by affecting processes like lipid retention in the vessel wall and inflammation, there have not been many in vitro disease models that include the effects of CS-remodeling that occurs in the body In our work, we demonstrated that enriching the TEBV extracellular matrix with pathological levels of CS leads to an enhanced atherosclerotic response to treatment with modified low-density lipoprotein (LDL), including increased VCAM expression, a marker of inflammation in endothelial cells, and increased white blood cell adhesion to the vessel wall.

In chapter 3, we tested the effects of BCAA treatment on endothelial cell and TEBV health. We cultured cells and TEBVs in a low-BCAA medium that reflected the BCAA levels that occur in human serum. While a few other studies have looked at the effects of BCAA on human vascular cells, they did not use physiologically relevant levels of BCAA in their untreated controls, and used much higher levels of BCAA doses to test their effects. In our studies, we found that BCAA affect several key processes related to endothelial health, inducing oxidative stress in the mitochondria, inducing increased expression of redox-balancing enzymes, and slowing autophagy. This was consistent with results in TEBV experiments, where we saw that BCAA cooperate with another pro-atherosclerotic agent, oxidized LDL, to induce vasodilation dysfunction and increased white blood cell adhesion to the vessel wall.

In chapter 4, we evaluated the hypothesis that slowing BCAA catabolism is sufficient to induce buildup of BCAA in vascular cells, leading to an atherosclerotic phenotype. To slow BCAA catabolism, we used a dCas9-KRAB construct to repress the gene PPM1K. PPM1K plays a critical regulatory role in modulating BCAA levels in the cell by activating the rate-limiting enzyme in the BCAA metabolic pathway and stimulating BCAA breakdown. We found that repressing PPM1K effectively alters the active state of its target enzyme, BCKDH, and increases glutamine and serine levels in iPSC-derived endothelial cells. In TEBVs, we found that PPM1K repressed-endothelium induces a differential response to oxLDL treatment in causing vasodilation dysfunction, compared to the vehicle control. Thus, there may be a role for BCAA metabolism in enhancing an atherosclerotic phenotype induced by other pro-atherosclerotic factors.

In summary, we determined that BCAA can contribute to an atherosclerotic phenotype, specifically by affecting endothelial cell health. This conclusion is supported by our observations that BCAA affect several key molecular and functional markers of endothelial health, including mitochondrial oxidative stress, autophagy, vasodilation function, and white blood cell adhesion to the endothelium. Importantly, in TEBVs, the presence of other pro-inflammatory factors, such as oxLDL, enhanced these effects. Future research should aim to identify which of these processes may be a suitable target to interrupt the atherosclerotic risk of BCAA.

Rheumatoid Arthritis (RA) is a chronic inflammatory auto-immune disease typically involving the joints, mainly the diarthrodial joint and generally starts between the age of 30 and 60 in women and somewhat later in life in men. It is the most common inflammatory arthritis and about one percent of the population is affected by RA. A complex interaction between various genetic and environmental factors lead to the development of the disease, though the specific cause of RA is not known. The goal of this study is to characterize the maturation of skeletal muscle bundles made with myoblasts isolated from RA patients and compare it with maturation of age-matched controls. Moreover, the engineered myobundles were treated with pro-inflammatory cytokines to assess their effect on the bundle maturation and to replicate the pro-inflammatory phenotype of RA.

Myobundles were prepared with human skeletal muscle (HSkM) samples obtained from young controls, age-matched controls and RA patients through biopsy of vastus lateralis muscle (biopsy of hamstring muscle was taken for young controls). We measured nuclei count, cross-sectional area, Myogenin count, Sarcomeric alpha-actinin (SAA) positive area and the myofiber diameter for each time course studies and cytokine treated bundles.

Contrary to our expectations, the time course study did not indicate significant reduction in fiber formation. This may be due to the effect of medications taken by the RA patient which might be helping the muscle function. Another possible reason might be that the cells could have regained their normal function once they were taken out from the inflammatory environment induced by the pro-inflammatory cytokines. Yet another possible reason may be that the time course considered may not be enough to access changes in the maturation and a longer time period may be required.

We then moved forward to replicate the disease pro-inflammatory phenotype by carrying out cytokine treatments on the engineered myobundles. IFNγ, IFNγ+GMCSF, TNFα+GMCSF and IFNγ+TNFα+GMCSF were chosen for the cytokine treatments. According to our results, the cross-sectional area, nuclei count/CSA, MyoG count/CSA, MyoG/Nuclei count, SAA+ area and the myofiber diameter each decreased with cytokine treatments indicating that the cytokines may indeed affect the regeneration ability of skeletal muscle cells.

The results from cytokines treatment studies indicate that cytokines do play a role in disease development and progression. A longer time course study say for up to 10 days or more post differentiation, more patient data regarding the disease severity and medications might also be helpful in further investigation.

Muscle fibrosis is caused by muscle injury, dystrophy, sarcopenia, and rheumatoid arthritis. This condition is characterized by hardening and scarring, which impairs contractile muscle function. To understand how fibrotic disease affects muscular function, we created a model of human skeletal muscle fibrosis using three-dimensional engineered skeletal muscle (myobundles). Furthermore, to investigate the effect of skeletal muscle fibrosis on the vascular system, we integrated the fibrotic skeletal muscle with tissue engineered blood vessels. Treating myobundles with Transforming Growth Factor β1 (TGF-β1) reproduced key characteristics of fibrotic skeletal muscle including reduced contractile force, disrupted contractile protein organization, increased stiffness, and expression of profibrotic genes. Treatment with a selective inhibitor (SB525334) of TGF-β1 receptor (ALK5, TGF-βRI) increased contractile function and decreased ECM deposition, consistent with animal studies in the literature. We also observed endogenous secretion of TGF-β1 in our myobundles which is of novel biological significance. siRNA knockdown of TGF-β1 increased contractile force. Testing anti-fibrotic drug Nintedanib in this model, showed an increase in tetanus force production in 2 out of 3 donors and reduction of pro-fibrotic ECM accumulation of collagen 1 and fibronectin. Western blot analysis of Nintedanib also providence evidence of its inhibition of TGF-β1 signaling by the reduction of phosphorylated Smad2/3. Repositioned anti-fibrotic drug Suramin treatment of fibrotic myobundles resulted in increase of tetanus force production in all three donors and reduction of pro-fibrotic ECM accumulation of collagen 1 and fibronectin. Suramin’s influence on TGF-β1 signaling in our system was found not to be as targeted as Nintedanib as there was only reduction in Smad3 phosphorylation and not Smad2 phosphorylation. Anti-fibrotic drug testing in our model was also able to wean out donor specific sensitivity to the drugs with donor 3. Skeletal myobundles were integrated with Tissue Engineered Blood Vessels (TEBVs) to identify the effect of skeletal muscle fibrosis on blood vessels or the human vasculature. Integrated TEBVs with 5 ng/ml TGF-β1 dosed myobundles showed reduced function, increased mesenchymal markers such as vimentin and alpha smooth muscle actin, and increased endothelial cell inflammation. Our results suggest a detrimental effect of skeletal muscle fibrosis on blood vessels and show an interaction between the skeletal muscle fibrosis and the human vasculature This model provides a platform to study skeletal muscle fibrosis alone or its effect on the vasculature and allows for testing anti-fibrotic drugs and assessing myobundle function along with disease influence on human vasculature.

There is a scarcity of knowledge regarding the cardiovascular effects of low dose ionizing radiation (IR) such as the one experienced during medical tests, radiation therapy or space travel. This is becoming more of a pressing problem given the enormous increase in radiation exposure by the average American today, and the renewed interest in deep space travel. Multiple epidemiologic studies suggest a higher rate of delayed cardiovascular related morbidity and mortality after low dose acute radiation exposure. These studies are significantly limited by a number of confounders such as cancer comorbidity, poor follow up, and largely estimated radiation doses that might not be accurate. These limitations are also seen in studies on the effect of space radiation on long term cardiovascular mortality and accelerated atherosclerosis. Animal studies have been used to simulate the effect of terrestrial and space radiation scenarios on cardiac function. These studies have led to conflicting conclusions, and had important challenges related to methods of assessment of cardiac injury. Furthermore, available studies to date had limited follow up times, and no study has evaluated the effects of more complex radiation scenarios that are likely to be experienced in space such as Galactic Cosmic Rays (GCRs). Our overall hypothesis is that IR is associated with early damage to healthy cardiomyocytes and vascular cells that eventually leads to long term cardiovascular dysfunction.

In the first part of this work, we hypothesize that IR is associated with a delayed cardiovascular derangement phenotype late after initial exposure. To test this hypothesis, we use a mouse animal model to study the effect of different radiation scenarios on cardiovascular function. Animals were exposed to one of the following (a) Gamma Rays (50-200 cGy), (b) 56Fe (15-50 cGy), (c) 16O (15-50cGy) heavy ions, and (d) 150cGy Galactic Cosmic Rays all using the particle accelerator at Brookhaven National Labs (BNL). They were then followed up for 9-12 months, and underwent cardiac MRI, pressure volume loop assessments, transthoracic echocardiograms and other histological evaluations. These studies revealed that GCR exposed animals had a clinically meaningful decline in their cardiac function, with a significant change in their arterial elastance. These findings were further confirmed on histology with their aortas demonstrating elastic fiber destruction and disorganization.

These animal studies however could not fully differentiate between a primary cardiac injury or a secondary cardiac response to a primary vascular injury to the aorta. Our hypothesis for the second part of this work, was that IR is associated with a unique and differentiated injury to cardiomyocytes that contributes to the previously seen cardiovascular phenotype. We therefore conducted additional experiments on isolated rat ventricular cardiomyocyte in collaboration with the Bursac lab. These studies used both 2D cultured cells, as well as a novel cardiac patch system both exposed to Gamma rays and X-rays (0.1-2 Gy). These cells underwent proteomics analysis, as well as a number of different biological and functional assays. While the acute functional effect of these radiation doses on cardiomyocytes was small, these irradiated cells produced a significant amount of reactive oxygen species and exhibited a large effect of radiation on mitochondrial related proteins, including elements of oxidative phosphorylation. We also noted a number of different pathways involved at different doses of radiation. This was an important finding, given that despite no changes in early cell death, the effect of these important proteomics changes on long term cardiac function maybe important.

Finally, for the last part of this dissertation, our hypothesis was that IR uniquely affects endothelial cells (ECs) and smooth muscle cells (SMCs) by inducing early senescence that is primarily due to over production of mitochondrial specific reactive oxygen species. To test this hypothesis, we use primary coronary artery endothelial cells, primary human aortic endothelial cells and primary coronary artery smooth muscle cells. These relevant cell types were then examined for their response to a single dose of radiation exposure. Given the previous findings of important mitochondria involvement even at low radiation doses, we used a novel mitochondrial specific ROS scavenger, that blocks the release of mROS, mito-TEMPO. Cells treated with mito-TEMPO had a significant decrease in observed cellular senescence an important hallmark indicator of cellular dysfunction. This strategy might have a potential therapeutic role in the prolonged cardiovascular effects of radiation exposure.

In summary, data generated in this dissertation supports the overall hypothesis that IR is associated with long term cardiovascular dysfunction that can be explained by early injury to cardiac, endothelial and smooth muscle cells. Galactic Cosmic Rays appear to significantly effect long term cardiovascular function, which has important implications on deep space travel. This effect is likely multifactorial, involving a number of organs, including the aorta. Cardiomyocytes, despite being resilient to death from radiation as compared to other cells types, appear to undergo a number of proteomics alterations after low dose radiation exposure, with significant involvement of the mitochondrial machinery. Finally, human vascular ECs and SMCs are highly sensitive to radiation exposure, and strategies that target mitochondrial specific ROS production might play an important role in mitigating the long-term vascular effects after radiation exposure.

The fatty acid linoleic acid has been identified as a potential mediator of atherosclerotic plaque development. Treatment of monocytes with linoleic acid leads to an increase in monocyte adhesion to endothelial cells under flow conditions; however, the mechanisms through which linoleic acid affect monocyte adhesion remain unclear. Using a combination of micropipette aspiration techniques and fluorescent microscopy, I tested the hypothesis that linoleic acid increases membrane tether formation between monocytes and endothelial cells.

Treatment of U937 monocytes with free linoleic acid or albumin-bound linoleic acid reduced the cortical tension of the monocytes. The effects of albumin-bound linoleic acid on the membrane were governed by the exchange of linoleic acid from albumin to the membrane and by the removal of fatty acids from the membrane by fatty acid binding sites on albumin.

The frequency of tether formation between U937 monocytes and TNF-α stimulated HUVECs increased following treatment with free linoleic acid or albumin-bound linoleic acid. The increase in tether frequency was not due to an increase in monocyte deformability or adhesion receptor expression. Tether extraction occurred primarily through E-selectin. Treatment with free linoleic acid increased the localization of E-selectin to clathrin-coated pits suggesting an increase in the formation of nanoclusters of E-selectin on HUVECs. The increase in tether frequency was blocked by the U73122 phospholipase C inhibitor indicating that linoleic acid increased monocyte adhesion through a phospholipase C mediated mechanism.

Treatment with free linoleic acid did not affect the threshold force for tether extraction or the effective viscosity of tethers extracted from HUVECs, but it decreased the threshold force for tether extraction from U937 monocytes and increased the effective tether viscosity. Treatment with U73122 blocked the reduction in the threshold force indicating that linoleic acid affected the regulation of the membrane adhesion energy through the hydrolysis of PIP2 by phospholipase C.

The results of the study indicated that linoleic acid promoted membrane tether formation by increasing E-selectin bond formation and reducing the adhesion energy between the U937 plasma membrane and the actin cytoskeleton through the hydrolysis of PIP2 by phospholipase C.

Advances in tissue engineering have led to the development of 3D biomimetic models of human skeletal muscle (myobundles), which have a wide range of applications in regenerative medicine, predictive drug testing and toxicology screening, and development of therapeutics for muscular disorders. Statins are the most commonly used medications to lower cholesterol and prevent cardiovascular diseases. Long term treatment using statins may lead to musculoskeletal side effects in the form of myopathy, myalgia or rhabdomyolysis. Currently, there are no reliable diagnostic measures of statin-induced myopathy since only some of the individuals complaining of the symptoms of statin myopathy exhibit elevated creatine kinase levels. This study examined if the engineered human skeletal myobundles using cells derived from patients with a history of statin induced myopathy exhibit statin-dependent defects in muscle physiology when exposed to varying concentrations of statins in vitro. Utilizing a microphysiological system for skeletal muscle, that uses patient-derived tissue to form engineered myobundles, to recapitulate the organization and function of native muscle is a novel way to understand the functional changes within individuals that experience statin induced myopathy. To create myobundles, myoblasts were encapsulated in a matrigel/fibrin matrix. The myobundles were cultured in 3D human growth media for 4 days and shifted to low amino acid differentiation media for another 4 days and then dosed for 5 days with varying concentrations of statins in low amino acid differentiation media. These were then force tested to see if there is a difference in twitch, tetanus and fatigue force response between different statin types, concentration levels, case and donor and dose exposure periods. A repeated measures multiple linear regression of the raw tetanus force versus statin dose, myopathy or control, and type of statin revealed a negative relationship between tetanus force response and statin concentration and myopathy. It was also seen that force production for the myopathy donors dosed with simvastatin when compared to their control pair was much lower which can be due to muscle weakness in these donors. In addition, immunofluorescence of the myobundles showed there is a structural difference in myotubule formation between the myopathy donors and control donors which can be correlated to force production. The donors that exhibited fewer myotubules or those that had a frayed appearance and lacked structural definition had lower force production. Overall, myobundles prepared with myoblasts from the myopathy and control donors exhibited functional changes associated with reported statin myopathy, while absence of myofibers or degradation of myofibers was associated with very low levels of force production.

Tissue-engineered skeletal muscle myobundles offer a promising approach for developing a human in vitro model of healthy and diseased muscle for drug development and testing. Their three-dimensional structure offers a better model of the organization of native skeletal muscle than monolayer culture does, and their amenability to an array of functional measures provides a multifaceted account of the tissue’s health. One such functional measure is the metabolic state of the myobundle, which in an in vitro model of healthy skeletal muscle should reflect the metabolism of native muscle. However, skeletal muscle cultured in vitro is exposed to artificially high levels of nutrients meant to promote cell growth, and it exhibits altered glucose uptake, with high rates of glycolysis and a dampened insulin response. Inflammation is closely linked with skeletal muscle metabolic dysfunction, and the field could benefit from a human tissue-engineered myobundle model of inflammation to elucidate mechanisms of disease progression and to examine drug safety and efficacy in inflamed muscle.

We first characterized the glucose uptake and insulin response of tissue-engineered myobundles in the basal state and in response to treatment with metformin and an HDAC inhibitor. We then imparted greater physiological relevance to the system via altered culture conditions and examined the impact on myobundle metabolic and contractile function. Finally, we validated the ability of pro-inflammatory cytokine exposed-myobundles to recapitulate key aspects of inflammation-mediated skeletal muscle dysfunction.

We found that myobundles exhibit insulin sensitivity similar to that of in vivo skeletal muscle, but insulin responsiveness is substantially lower, in accordance with other in vitro studies. Metformin treatment stimulated a robust increase in basal glucose uptake, and treatment with the HDAC inhibitor 4-PBA enhanced myobundle contractile function and insulin responsiveness. We showed that altering myobundle culture conditions to provide more physiologic nutrient availability was sufficient to metabolically reprogram the myobundles to a less glycolytic state. To model an inflammation-mediated disease state, we exposed myobundles to pro-inflammatory cytokines and found that the inflamed myobundles exhibited contractile dysfunction, a robust secretion of pro-inflammatory cytokines, and increased basal glucose uptake while still retaining a functional response to metformin treatment. Overall, this work validates the suitability of a tissue-engineered skeletal muscle model for detecting metabolic perturbations mediated by drug treatment, nutrient availability, and inflammatory conditions.

High shear stresses and shear rates in left ventricular assist devices (LVADs) make endothelialization of the LVAD difficult and likely contribute to cleavage of large von Willebrand factor multimers and resulting bleeding problems in patients. To better understand shear in a centrifugal LVAD, flow was simulated using finite volume and computational fluid dynamics (CFD) analysis. The k-ω model simulated turbulence and sliding meshes were used to model the movement of the impeller. CFD results showed high-shear backflows in the radial gap between the impeller and the volute wall, but residence times in this region were under 5ms. It is unclear if this is sufficient to cleave VWF, and more study is necessary to determine if other areas in the LVAD have potential for VWF cleavage. Although the walls near the outlet experience low shear stress and may be good candidates for endothelialization, shear stresses above 20-30Pa on all other walls of the pump make the possibility of endothelial cell growth elsewhere in the LVAD unlikely. An LVAD designed specifically to have low shear may be a better candidate for endothelialization.

hCB-ECs show varying morphology under co-culture conditions. They are known to form networks when co-cultured on matrices like Matrigel, collagen gels and SMCs. Optimizing the co-culture model for the formation of networks can enable better understanding of angiogenesis and can aid in the area of tissue engineered organs while creating a model with no networks can help in producing a smooth layer of cells for tissue engineered blood vessels. Additionally, the study of networks on smooth muscle cell surfaces gives a better approach to understanding the in vivo phenomena.

The main goal of this study was to identify conditions that would support the formation of networks and to study the gene level alterations in the hCB-EC cells between co-culture and monoculture during the formation and absence of networks. To study the morphological changes co-cultures were setup by varying the hCB-EC densities at 26,316 cells/cm2, 52,632 cells/cm2, 80,000 cells/cm2 and 105,263 cells/cm2 on SMCs. Lower seeding densities of hCB-ECs led to network formations while a confluent layer was observed at the highest density. Medium components were altered to identify factors which contributed to network formation and it was found that absence of VEGF led to delayed cell migration and network formation while absence of heparin produced sparser networks in co-culture. Microarray analysis using four different hCB-EC sources plated at the highest and lowest densities resulted in higher expression of ECM remodeling and endothelial cell migration genes consistent to the low density conditions were networks were abundant while high density conditions expressed downregulation of cell cycle associated genes.