Browsing by Subject "Endothelial cell"

The vasculature has a crucial function in normal physiology, enabling the transport of oxygen and nutrients to cells throughout the body. In turn, endothelial cells, which form the inner-most lining of blood vessels, are key regulators of vascular function. In addition to forming a barrier which separates the circulation from underlying tissues, endothelial cells respond to diverse extracellular cues and produce a variety of biologically-active mediators in order to maintain vascular homeostasis. Disruption of normal vascular function is a prominent feature of a variety of pathological conditions. Thus, elucidating the signaling pathways regulating endothelial function is critical for understanding the role of endothelial cells in both normal physiology and pathology, as well as for potential development of therapeutic interventions.

In this dissertation, we use a combination of pharmacological inhibition and knockdown studies, along with generation of endothelial conditional knockout mice, to demonstrate an important role of the Abelson (Abl) family of non-receptor tyrosine kinases (Abl and Arg) in vascular function. Specifically, loss of endothelial expression of the Abl kinases leads to late-stage embryonic and perinatal lethality in conditional knockout mice, indicating a crucial requirement for Abl/Arg kinases in normal vascular development and function. Endothelial Abl/Arg-null embryos display focal regions of vascular loss and tissue damage, as well as increased endothelial cell apoptosis. An important pro-survival function for the Abl kinases is further supported by our finding that either microRNA-mediated Abl/Arg depletion or pharmacological inhibition of the Abl kinases increases endothelial cell susceptibility to stress-induced apoptosis in vitro. The Abl kinases are activated in response to treatment with the pro-angiogenic growth factors vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF). We show that both VEGF- and bFGF-mediated endothelial cell survival is impaired following Abl kinase inhibition.

These studies have uncovered a previously unappreciated role for the Abl kinases in the regulation of the angiopoietin/Tie2 signaling pathway, which functions to support endothelial cell survival and vascular stability. Loss of Abl/Arg expression leads to reduced mRNA and protein levels of the Tie2 receptor, resulting in impaired activation of intracellular signaling pathways by the Tie2 ligand angiopoietin-1 (Angpt1), as well as decreased Angpt1-mediated endothelial cell survival following serum-deprivation stress. Notably, we found that the Abl kinases are activated following Angpt1 stimulation, suggesting a unique dual role for Abl and Arg in Angpt/Tie2 signaling, potentially modulating Tie2 downstream signaling responses, as well as regulating Tie2 receptor expression.

Further, we show an important contribution of the Abl family kinases to the regulation of endothelial permeability responses both in vitro and in vivo. The Abl kinases are activated in response to a diverse group of permeability-inducing factors, including VEGF and the inflammatory mediators thrombin and histamine. We show that inhibition of Abl kinase activity, using either the ATP-competitive inhibitor imatinib or the allosteric inhibitor GNF-2, protects against disruption of endothelial barrier function by the permeability-inducing factors in vitro. VEGF-induced vascular permeability similarly is decreased in conditional knockout mice lacking endothelial Abl expression, as well as following treatment with Abl kinase inhibitors in vivo. Mechanistically, we show that loss of Abl kinase activity is accompanied by activation of the barrier-stabilizing GTPases (guanosine triphosphatases) Rac1 and Rap1, as well as inhibition of agonist-induced Ca2+ mobilization and generation of acto-myosin contractility.

Taken together, these results demonstrate involvement of the Abl family kinases in the regulation of endothelial cell responses to a broad range of pro-angiogenic and permeability-inducing factors, as well as a critical requirement for the endothelial Abl kinases in normal vascular development and function in vivo. These findings have implications for the clinical use of Abl kinase inhibitors.

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.

The adaptive response of vascular endothelial cells to shear stress alteration induced by global hemodynamic changes is an essential component of normal endothelial physiology in vivo; and an understanding of the transient regulation of endothelial phenotype during adaptation will advance our understanding of endothelial biology and yield new insights into the mechanism of atherogenesis. The objective of this study was to characterize the adaptive response of arterial endothelial cells to acute increases in shear stress magnitude and frequency in well-defined in vitro settings. Porcine endothelial cells were preconditioned by a basal level shear stress of ±15dynes/cm^2 at 1 Hz for 24 hours, and an acute increase in shear stress magnitude (30 ±15 dynes/cm^2) or frequency (2 Hz) was then applied. Endothelial permeability to bovine serum albumin was measured and gene expression profiling was performed using microarrays at multiple time points during a period of 6 hours after the shear stress alteration. The instantaneous endothelial permeability was found to increase rapidly in response to the acute increase in shear stress magnitude. Endothelial permeability nearly doubled after 40 minutes exposure to the elevated shear magnitude, and then decreased gradually. However, less dependency of endothelial permeability on shear stress frequency was observed. Endothelial permeability increased slowly from 120 minutes to 6 hours after exposure to the elevated shear frequency, but the increase was not statistically significant and was relatively small (1.2 fold increase at 6 hours). The transcriptomics studies identified 86 genes that were sensitive to the elevated shear magnitude and 37 genes sensitive to the elevated frequency. A significant number of the identified genes are previously unknown as sensitive to shear stress. The acute increase in shear magnitude promoted the expression of a group of anti-inflammatory and anti-oxidative genes; while the acute increase in shear frequency upregulated a set of cell-cycle regulating genes and angiogenesis genes. The adaptive response of global gene expression profile to the elevated shear magnitude is found to be triphasic, consisting of an induction period, an early adaptive response (ca. 45 minutes) and a late remodeling response. However, no apparent temporal regulation pattern of global gene expression was found during the adaptation to the elevated shear frequency. The results from this dissertation suggest that endothelial cells exhibit a specific phenotype during the adaptive response to changes in shear stress; and the transient phenotype is different than that of fully-adapted endothelial cells and may alter arterial atherosusceptibility.

Approximately half of all cancer patients will receive radiation as part of their treatment regimen in either a palliative or curative setting. Conventionally, radiation therapy has been administered to patients in small 1.8-2 Gy daily fractions over the span of 1 to 2 months. However, advances in treatment planning and radiation delivery methods have enabled tumors to be irradiated with a small number of large radiation doses between 15 and 24 Gy. This innovative radiation delivery method has been employed for the treatment of various cancers, resulting in improved rates of local control. However, the mechanism mediating enhanced tumor eradication with high dose radiation therapy remains elusive. Some studies have suggested that the tumor vasculature is functionally damaged or destroyed following high doses of radiation, resulting in a second wave of tumor cell killing due to the depletion of nutrients. Other studies suggest that although high doses of radiation may disrupt the tumor vasculature, this phenotype is not a critical regulator of the enhanced rates of local control obtained with high dose radiation therapy. A better understanding of the mechanisms by which high radiation dose per fraction improves the efficacy of radiation therapy will help to develop strategies to improve patient outcomes.

To dissect the critical cellular target(s) of radiation therapy that regulates tumor response, we utilized genetically engineered mouse models of soft tissue sarcoma, non-small cell lung cancer, and brainstem glioma to manipulate the radiosensitivity of various cellular compartments. Dual recombinase technology enabled the deletion of the DNA damage response gene Atm in tumor endothelial cells. In primary sarcomas, radiosensitization of the tumor vasculature by disruption of ATM signaling resulted in improved tumor growth delay, but not rates of local control in response to radiation. The radiosensitization of endothelial cells did not improve tumor radiation responses in primary models of lung cancer and brainstem glioma. In stark contrast, deletion of Atm within the tumor parenchymal cells specifically resulted in improved radiation therapy outcomes in all three mouse models. Interestingly, loss of Atm was only able to improve tumor outcome after radiotherapy in brainstem gliomas lacking p53 which has potential significance for the translation of ATM inhibitors into the clinic. Together, these results identify tumor parenchymal cells, rather than endothelial cells, as the critical targets that promote tumor response to radiation therapy.