Browsing by Subject "Endothelial cells"
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Item Open Access A PK2/Bv8/PROK2 antagonist suppresses tumorigenic processes by inhibiting angiogenesis in glioma and blocking myeloid cell infiltration in pancreatic cancer.(2011) Curtis, Valerie ForbesIn many cancer types, infiltration of bone marrow-derived myeloid cells in the tumor microenvironment is often associated with enhanced angiogenesis and tumor progression, resulting in poor prognosis. The polypeptide chemokine PK2 (Bv8) regulates myeloid cell mobilization from the bone marrow, leading to activation of angiogenesis as well as accumulation of macrophages and neutrophils in the tumor site. Neutralizing antibodies against PK2 display potent anti-tumor efficacy, illustrating the potential of PK2-antagonists as therapeutic agents for the treatment of cancer. However, antibody-based therapies can be too large to treat certain diseases and too expensive to manufacture while small molecule therapeutics are not prohibitive in these ways. In this study, we demonstrate the anti-tumor activity of a small molecule PK2 antagonist, PKRA7, in the contexts of glioblastoma and pancreatic cancer xenograft tumor models. In the highly vascularized glioblastoma, PKRA7 decreased blood vessel density while increasing necrotic areas in the tumor mass. Consistent with the anti-angiogenic activity of PKRA7 in vivo, this compound effectively reduced PK2-induced microvascular endothelial cell branching in vitro. For the poorly vascularized pancreatic cancer, the primary anti-tumor effect of PKRA7 is mediated by the blockage of myeloid cell migration and infiltration. At the molecular level, PKRA7 inhibits PK2-induced expression of several pro-migratory chemokines and chemokine receptors in macrophages. Combining PKRA7 treatment with standard chemotherapeutic agents resulted in enhanced effects in xenograft models for both glioblastoma and pancreatic tumors. Taken together, our results indicate that the anti-tumor activity of PKRA7 can be mediated by distinct mechanisms that are relevant to the pathological features of the specific type of cancer. This small molecule PK2 antagonist holds the promise to be further developed as an effective agent for combinational cancer therapy.Item Open Access Characterizing the Role of the Previously Undescribed Protein Caskin2 in Vascular Biology(2016) Mueller, Sarah BethMaintenance of vascular homeostasis is an active process that is dependent on continuous signaling by the quiescent endothelial cells (ECs) that line mature vessels. Defects in vascular homeostasis contribute to numerous disorders of significant clinical impact including hypertension and atherosclerosis. The signaling pathways that are active in quiescent ECs are distinct from those that regulate angiogenesis but are comparatively poorly understood. Here we demonstrate that the previously uncharacterized scaffolding protein Caskin2 is a novel regulator of EC quiescence and that loss of Caskin2 in mice results in elevated blood pressure at baseline. Caskin2 is highly expressed in ECs from various vascular beds both in vitro and in vivo. When adenovirally expressed in vitro, Caskin2 inhibits EC proliferation and migration but promotes survival during hypoxia and nutrient deprivation. Likewise, loss of Caskin2 in vivo promotes increased vascular branching and permeability in mouse and zebrafish models. Caskin2 knockout mice are born in normal Mendelian ratios and appear grossly normal during early adulthood. However, they have consistently elevated systolic and diastolic blood pressure at baseline and significant context-dependent abnormalities in systemic metabolism (e.g., body weight, fat deposition, and glucose homeostasis). Although the precise molecular mechanisms of these effects remain unclear, we have shown that Caskin2 interacts with several proteins known to have important roles in endothelial biology and cardiovascular disease including the serine/threonine phosphatase PP1, the endothelial receptor Tie1, and eNOS, which is a critical regulator of vascular homeostasis. Ongoing work seeks to further characterize the functions of Caskin2 and its mechanisms of action with a focus on how Caskin2-mediated regulation of endothelial phenotype relates to its systemic effects on cardiovascular and metabolic function.
Item Open Access Dissecting Tumor Response to Radiation Therapy Using Genetically Engineered Mouse Models(2015) Moding, Everett JamesApproximately 50% of all patients with cancer receive radiation therapy at some point during the course of their illness. Despite advances in radiation delivery and treatment planning, normal tissue toxicity often limits the ability of radiation to eradicate tumors. The tumor microenvironment consists of tumor cells and stromal cells such as endothelial cells that contribute to tumor initiation, progression and response to therapy. Although endothelial cells can contribute to normal tissue injury following radiation, the contribution of stromal cells to tumor response to radiation therapy remains controversial. To investigate the contribution of endothelial cells to the radiation response of primary tumors, we have developed the technology to contemporaneously mutate different genes in the tumor cells and stromal cells of a genetically engineered mouse model of soft tissue sarcoma. Using this dual recombinase technology, we deleted the DNA damage response gene Atm in sarcoma and heart endothelial cells. Although deletion of Atm increased cell death of proliferating tumor endothelial cells, Atm deletion in quiescent endothelial cells of the heart did not sensitize mice to radiation-induced myocardial necrosis. In addition, the ATM inhibitor NVP-BEZ235 selectively radiosensitized primary sarcomas, demonstrating a therapeutic window for inhibiting ATM during radiation therapy. Sensitizing tumor endothelial cells to radiation by deleting Atm prolonged tumor growth delay following a non-curative dose of radiation, but failed to increase local control. In contrast, deletion of Atm in tumor parenchymal cells increased the probability of tumor eradication. These results demonstrate that tumor parenchymal cells rather than endothelial cells are the critical targets that regulate tumor eradicaiton by radiation therapy.
Item Open Access Interleukin-9 mediates chronic kidney disease-dependent vein graft disease: a role for mast cells.(Cardiovasc Res, 2017-11-01) Zhang, Lisheng; Wu, Jiao-Hui; Otto, James C; Gurley, Susan B; Hauser, Elizabeth R; Shenoy, Sudha K; Nagi, Karim; Brian, Leigh; Wertman, Virginia; Mattocks, Natalie; Lawson, Jeffrey H; Freedman, Neil JAims: Chronic kidney disease (CKD) is a powerful independent risk factor for cardiovascular events, including vein graft failure. Because CKD impairs the clearance of small proteins, we tested the hypothesis that CKD exacerbates vein graft disease by elevating serum levels of critical cytokines that promote vein graft neointimal hyperplasia. Methods and results: We modelled CKD in C57BL/6 mice with 5/6ths nephrectomy, which reduced glomerular filtration rate by 60%, and we modelled vein grafting with inferior-vena-cava-to-carotid interposition grafting. CKD increased vein graft neointimal hyperplasia four-fold, decreased vein graft re-endothelialization two-fold, and increased serum levels of interleukin-9 (IL-9) five-fold. By quantitative immunofluorescence and histochemical staining, vein grafts from CKD mice demonstrated a ∼two-fold higher prevalence of mast cells, and a six-fold higher prevalence of activated mast cells. Concordantly, vein grafts from CKD mice showed higher levels of TNF and NFκB activation, as judged by phosphorylation of NFκB p65 on Ser536 and by expression of VCAM-1. Arteriovenous fistula veins from humans with CKD also showed up-regulation of mast cells and IL-9. Treating CKD mice with IL-9-neutralizing IgG reduced vein graft neointimal area four-fold, increased vein graft re-endothelialization ∼two-fold, and reduced vein graft total and activated mast cell levels two- and four-fold, respectively. Treating CKD mice with the mast cell stabilizer cromolyn reduced neointimal hyperplasia and increased re-endothelialization in vein grafts. In vitro, IL-9 promoted endothelial cell apoptosis but had no effect on smooth muscle cell proliferation. Conclusion: CKD aggravates vein graft disease through mechanisms involving IL-9 and mast cell activation.Item Open Access Mechanisms by which p53 Regulates Radiation-induced Carcinogenesis and Myocardial Injury(2012) Lee, ChangLungRadiation therapy can cause acute toxicity and long-term side effects in normal tissues. Because part of the acute toxicity of radiation is due to p53-mediated apoptosis, blocking p53 during irradiation can protect some normal tissues from acute radiation injury and might improve the therapeutic ratio of radiation therapy. However, the mechanisms by which p53 regulates late effects of radiation are not well understood. Here, I utilized genetically engineered mouse models to dissect the role of p53 in regulating two of the most clinically significant late effects of radiation: radiation-induced carcinogenesis and radiation-induced myocardial injury.
It has been well characterized that mice with one allele of p53 permanently deleted are sensitized to radiation-induced cancer. Therefore, temporary inhibition of blocking p53 during irradiation could promote malignant transformation. Experiments with mice lacking functional p53 in which p53 protein can be temporarily restored during total-body irradiation (TBI) suggest that the radiation-induced p53 response does not contribute to p53-mediated tumor suppression. Here, I performed reciprocal experiments and temporarily turned p53 off during TBI using transgenic mice with reversible RNA interference against p53. I found that temporary knockdown of p53 during TBI not only ameliorated acute hematopoietic toxicity, but in both Kras wild-type and tumor-prone KrasLA1 mice also prevented lymphoma development. Mechanistic studies show that p53 knockdown during TBI improves survival of hematopoietic stem and progenitor cells (HSPCs), which maintains HSPC quiescence and prevents accelerated repopulation of surviving cells. Moreover, using an in vivo competition assay I found that temporary knockdown of p53 during TBI maintains the fitness of p53 wild-type HSPCs to prevent the expansion of irradiated mutant cells. Taken together, our data demonstrate that p53 functions during TBI to promote lymphoma formation by facilitating the expansion of irradiated HSPCs with adaptive mutations.
p53 functions in the heart to promote myocardial injury after multiple types of stress, including ischemic injury, pressure overload and doxorubicin-induced oxidative stress. However, how p53 regulates radiation-induced myocardial injury, which develops after radiation therapy, is not well understood. Here, I utilized the Cre-loxP system to demonstrate that p53 functions in endothelial cells to protect mice from myocardial injury after a single dose of 12 Gy or 10 daily fractions of 3 Gy whole-heart irradiation (WHI). Mice in which both alleles of p53 are deleted in endothelial cells succumbed to heart failure after WHI due to myocardial necrosis, systolic dysfunction and cardiac hypertrophy. Moreover, the onset of cardiac dysfunction was preceded by alterations in myocardial vascular permeability and density. Mechanistic studies using primary cardiac endothelial cells (CECs) irradiated in vitro indicate that p53 signals to cause a mitotic arrest and protects CECs against radiation-induced mitotic catastrophe. Furthermore, mice lacking the cyclin-dependent kinase inhibitor p21, which is a transcriptional target of p53, are also sensitized to myocardial injury after 12 Gy WHI. Together, our results demonstrate that the p53/p21 axis functions to prevent radiation-induced myocardial injury in mice. Our findings raise the possibility that when combining radiation therapy with inhibitors of p53 or other components of the DNA damage response that regulate mitotic arrest, patients may experience increased radiation-related heart disease.
Taken together, our results demonstrate crucial but distinct roles of p53 in regulating late effects of radiation: p53-mediated apoptosis promotes radiation-induced lymphomagenesis, but p53-mediated cell cycle arrest prevents radiation-induced myocardial injury. These findings indicate that p53 may generally play a protective role from radiation, particularly at high doses, in cells where p53 activation is uncoupled from the induction of the intrinsic pathway of apoptosis. Therefore, selectively inhibiting p53-mediated apoptosis may be a promising approach to ameliorate acute radiation toxicity without exacerbating late effects of radiation.
Item Open Access Umbilical Cord Blood Derived Endothelial Progenitor Cells: Isolation, Characterization, and Adhesion Potential in Vitro and in Vivo(2009) Brown, Melissa AnnThe number one cause of death in the industrialized world, atherosclerosis, can be treated through a variety of methods: angioplasty, stenting, vein graft bypass, synthetic grafts, and maybe one day tissue engineering vessels (TEBVs). The long term goal that motivated this research is the delivery of umbilical cord blood derived endothelial progenitor cells (CB-EPCs) to damaged arteries and possibly reducing the rate of re-occlusion by re-establishing a healthy, functional, intact endothelium. The proposed research tested the following hypotheses: (1) Mild trypsinization methods produces strong endothelial cell (EC) adhesion strength, (2) CB-EPCs are functionally similar to native ECs (specifically human aortic endothelial cells (HAECs)) and exhibit similar anti-thrombotic and anti-inflammatory behavior compared to HAECs, (3) CB-EPCs are capable of adhering to smooth muscle cells (SMCs) and extracellular matrix (ECM) proteins under flow conditions, (4) CB-EPCs can be used to prevent thrombosis in mice that have undergone vein bypass grafts through re-endothelialization of the vessel, and (5) CB-EPCs are capable of proliferating under flow conditions. In order to produce supraphysiological adhesion strengths of HAECs or CB-EPCs, the cells must be detached using 0.025% trypsin for 5 minutes prior to adhesion to adsorbed ECM proteins or SMCs. CB-EPCs have a high proliferation rate and express similar levels of important anti-thrombotic genes and inflammatory proteins compared to HAECs. CB-EPCs and HAECs produce similar levels of nitric oxide and alignment in the direction of flow when exposed to laminar shear stress for at least 24 hours. CB-EPCs are capable of adhering to many different substrates under flow conditions. The adhesion of CB-EPCs with response to shear stress appears to be biphasic and increases with shear stress up to 0.75 dyn/cm2 and then decreases above this value. CB-EPC adhesion is much greater than HAECs and EPCs isolated from peripheral blood (PB-EPCs) of healthy individuals, which can be related to their higher expression level of adhesion integrin α5β1 and their smaller size. When seeded onto FN coated plastic, CB-EPCs proliferated under flow conditions and had a much shorter doubling time than PB-EPCs and HAECs. Proliferation of CB-EPCs and HAECs on SMCs was limited. Further, Cb-EPCs formed network-like structures except when growth factors were removed and a shear stress of at least 5 dyne/cm2 was applied. To assess whether CP-EPCs could promote vessel repair in vivo, human CB-EPCs were injected into SCID mice that received a carotid interpositional vein grafts, resulting in 100% patency. In contrast, only 2 of the 8 saline injected mice had a patent vein graft 2 weeks post surgery. We found that CB-EPC injected mice had roughly 55% endothelialization compared to less than 20% for the patent saline controls, with CB-EPCs making up approximately 33% of this coverage. These results suggest that CB-EPCs could be used as a therapeutic method to prevent vessel re-occlusion in patients undergoing treatment for atherosclerosis.
Item Open Access Use of Human Blood-Derived Endothelial Progenitor Cells to Improve the Performance of Vascular Grafts(2011) Stroncek, JohnSynthetic small diameter vascular grafts fail clinically due to thrombosis and intimal hyperplasia. The attachment of endothelial cells (ECs) onto the inner lumen of synthetic small diameter vascular grafts can improve graft patency; however, significant challenges remain that prevent wide clinical adoption. These issues include difficulties in the autologous sourcing of ECs, the lack of attachment, growth and retention of the layer of ECs to the graft lumen, and the maintenance of an anti-thrombotic and anti-inflammatory profile by the layer of ECs.
This dissertation describes the isolation, characterization, and use of endothelial progenitor cells (EPCs) to improve the performance of small diameter vascular grafts. First, EPC isolation efficiency and expression of critical EC markers was compared between young healthy volunteers and patients with documented coronary artery disease (CAD). EPCs were isolated and expanded from patients with CAD and had a similar phenotype to EPCs isolated from healthy donors, and a control population of human aortic ECs. Second, we assessed the ability to enhance the anti-thrombotic activity of patient derived EPCs through the over expression of thrombomodulin (TM). In vitro testing showed TM-transfected EPCs had significantly increased production of key anti-thrombotic molecules, reduced platelet adhesion, and extended clotting times over untransfected EPCs. Finally, native and TM-transfected EPCs were seeded onto small diameter vascular grafts and tested for their ability to improve graft performance. EPCs sodded onto the lumen of small diameter ePTFE vascular grafts had strong adhesion and remained adherent during graft clamping and exposure to flow. TM-transfected EPCs improved graft anti-thrombotic performance significantly over bare grafts and grafts seeded with native EPCs. Based on these promising in vitro results, grafts were implanted bilaterally into the femoral arteries of athymic rats. Bare grafts and grafts with air removed clotted and had only 25% patency at 7 days. In contrast, graft sodded with native EPCs or TM-transfected EPCs had 87% and 89% respective patency rates. High patency rates continued with 28 day implant testing with EPC sodded grafts (88% Native; 75% TM). There were no significant differences in patency rates at 7 or 28 days between native and TM-transfected grafts. These in vivo data suggest patient blood-derived EPCs can be used to improve the performance of small diameter vascular grafts.