Urokinase-type Plasminogen Activator (uPA) Promotes Angiogenesis by Attenuating Proline-rich Homeodomain Protein (PRH) Transcription Factor Activity and De-repressing Vascular Endothelial Growth Factor (VEGF) Receptor Expression

Urokinase-type plasminogen activator (uPA) regulates angiogenesis and vascular permeability through proteolytic degradation of extracellular matrix and intracellular signaling initiated upon its binding to uPAR/CD87 and other cell surface receptors. Here, we describe an additional mechanism by which uPA regulates angiogenesis. Ex vivo VEGF-induced vascular sprouting from Matrigel-embedded aortic rings isolated from uPA knock-out (uPA(−/−)) mice was impaired compared with vessels emanating from wild-type mice. Endothelial cells isolated from uPA(−/−) mice show less proliferation and migration in response to VEGF than their wild type counterparts or uPA(−/−) endothelial cells in which expression of wild type uPA had been restored. We reported previously that uPA is transported from cell surface receptors to nuclei through a mechanism that requires its kringle domain. Intranuclear uPA modulates gene transcription by binding to a subset of transcription factors. Here we report that wild type single-chain uPA, but not uPA variants incapable of nuclear transport, increases the expression of cell surface VEGF receptor 1 (VEGFR1) and VEGF receptor 2 (VEGFR2) by translocating to the nuclei of ECs. Intranuclear single-chain uPA binds directly to and interferes with the function of the transcription factor hematopoietically expressed homeodomain protein or proline-rich homeodomain protein (HHEX/PRH), which thereby lose their physiologic capacity to repress the activity of vehgr1 and vegfr2 gene promoters. These studies identify uPA-dependent de-repression of vegfr1 and vegfr2 gene transcription through binding to HHEX/PRH as a novel mechanism by which uPA mediates the pro-angiogenic effects of VEGF and identifies a potential new target for control of pathologic angiogenesis

Inhibition of pathologic retinal neovascularization by α-defensins

Proliferative retinopathies, such as those complicating prematurity and diabetes, are major causes of blindness. A prominent feature of these retinopathies is excessive neovascularization, which is orchestrated by the hypoxia-induced vascular endothelial growth factor (VEGF) stimulating endothelial cells and the integrin-mediated adhesive interactions of endothelial cells with extracellular matrix components such as fibronectin (FN). Recently, we demonstrated that α-defensins interfere with α5β1–FN interactions and dependent endothelial cell functions. Here, α-defensins were studied in hypoxia-induced proliferative retinopathy. In vitro, α-defensins specifically inhibited α5β1-integrin–dependent migration of bovine retinal endothelial cells (BRECs) to FN, attenuated the VEGF-stimulated increase in endothelial permeability, and blocked BREC proliferation and capillary sprout formation in 3-dimensional fibrin-matrices. An up-regulation of β1-integrin and FN was observed in the retinal vessels in the mouse model of hypoxia-induced retinal angiogenesis. Systemic and local administration of α-defensins reduced retinal neovascularization by 45% and 60%, respectively, and this effect was comparable to the inhibitory effect of α5β1-blocking antibody. α-Defensins were detected in human diabetic retinas associated with normal retinal vessels but were absent from proliferative lesions. Together, these data show that α-defensins inhibit pathologic retinal neovascularization in vivo and may provide a clinically efficient strategy against proliferative retinopathies

Adrenomedullin Prevents Sex-Dependent Impairment of Autoregulation during Hypotension after Piglet Brain Injury through Inhibition of ERK MAPK Upregulation

Cerebrospinal fluid (CSF) adrenomedullin (ADM) levels are increased in female, but remain unchanged in male, piglets after fluid percussion injury (FPI) of the brain. Subthreshold vascular concentrations of ADM restore impaired hypotensive pial artery dilation after FPI more in males than females. Extracellular signal-related kinase (ERK) mitogen-activated protein kinase (MAPK) is upregulated and contributes to reductions in cerebral blood flow (CBF) after FPI. We hypothesized that ADM prevents sex-dependent impairment of autoregulation during hypotension after FPI through inhibition of ERK MAPK upregulation. FPI increased ERK MAPK more in males than in females. CBF was unchanged during hypotension in sham animals, was reduced more in males than in females after FPI during normotension, and was further reduced in males than in females during hypotension and after FPI. ADM and the ERK MAPK antagonist U 0126 prevented reductions in CBF during hypotension and FPI more in males than in females. Transcranial Doppler (TCD) blood flow velocity was unchanged during hypotension in sham animals, was decreased during hypotension and FPI in male but not in female pigs, and was ameliorated by ADM. Intracranial pressure (ICP) was increased after FPI more in male than in female animals. ADM blunted elevated ICP during FPI and hypotension in males, but not in females. ADM prevented reductions in cerebral perfusion pressure (CPP) during FPI and hypotension in males but not in females. The calculated autoregulatory index was unchanged during hypotension in sham animals, but was reduced more in males than females during hypotension and FPI. ADM prevented reductions in autoregulation during hypotension and FPI more in males than females. These data indicate that ADM prevented loss of cerebral autoregulation after FPI in a sex-dependent and ERK MAPK-dependent manner

Opposing effects of HNP1 (α-defensin-1) on plasma cholesterol and atherogenesis.

Atherosclerosis, the predominant cause of death in well-resourced countries, may develop in the presence of plasma lipid levels within the normal range. Inflammation may contribute to lesion development in these individuals, but the underlying mechanisms are not well understood. Transgenic mice expressing α-def-1 released from activated neutrophils develop larger lipid and macrophage-rich lesions in the proximal aortae notwithstanding hypocholesterolemia caused by accelerated clearance of α-def-1/low-density lipoprotein (LDL) complexes from the plasma. The phenotype does not develop when the release of α-def-1 is prevented with colchicine. However, ApoE-/- mice crossed with α-def-1 mice or given exogenous α-def-1 develop smaller aortic lesions associated with reduced plasma cholesterol, suggesting a protective effect of accelerated LDL clearance. Experiments were performed to address this seeming paradox and to determine if α-def-1 might provide a means to lower cholesterol and thereby attenuate atherogenesis. We confirmed that exposing ApoE-/- mice to α-def-1 lowers total plasma cholesterol and decreases lesion size. However, lesion size was larger than in mice with total plasma cholesterol lowered to the same extent by inhibiting its adsorption or by ingesting a low-fat diet. Furthermore, α-def-1 levels correlated independently with lesion size in ApoE-/- mice. These studies show that α-def-1 has competing effects on atherogenesis. Although α-def-1 accelerates LDL clearance from plasma, it also stimulates deposition and retention of LDL in the vasculature, which may contribute to development of atherosclerosis in individuals with normal or even low plasma levels of cholesterol. Inhibiting α-def-1 may attenuate the impact of chronic inflammation on atherosclerotic vascular disease

Direct interaction of the kringle domain of urokinase-type plasminogen activator (uPA) and integrin alpha v beta 3 induces signal transduction and enhances plasminogen activation

It has been questioned whether there are receptors for urokinase-type plasminogen activator (uPA) that facilitate plasminogen activation other than the high affinity uPA receptor (uPAR/CD87) since studies of uPAR knockout mice did not support a major role of uPAR in plasminogen activation. uPA also promotes cell adhesion, chemotaxis, and proliferation besides plasminogen activation. These uPA-induced signaling events are not mediated by uPAR, but mediated by unidentified, lower-affinity receptors for the uPA kringle. We found that uPA binds specifically to integrin alpha v beta 3 on CHO cells depleted of uPAR. The binding of uPA to alpha v beta 3 required the uPA kringle domain. The isolated uPA kringle domain binds specifically to purified, recombinant soluble, and cell surface alpha v beta 3, and other integrins (alpha 4 beta 1 and alpha 9 beta 1), and induced migration of CHO cells in an alpha v beta 3-dependent manner. The binding of the uPA kringle to alpha v beta 3 and uPA kringle-induced alpha v beta 3-dependent cell migration were blocked by homologous plasminogen kringles 1-3 or 1-4 (angiostatin), a known integrin antagonist. We studied whether the binding of uPA to integrin alpha v beta 3 through the kringle domain plays a role in plasminogen activation. On CHO cell depleted of uPAR, uPA enhanced plasminogen activation in a kringle and alpha v beta 3-depenclent manner. Endothelial cells bound to and migrated on uPA and uPA kringle in an alpha v beta 3-dependent manner. These results suggest that uPA binding to integrins through the kringle domain plays an important role in both plasminogen activation and uPA-induced intracellular signaling. The uPA kringle-integrin interaction may represent a novel therapeutic target for cancer, inflammation, and vascular remodeling

Proteomic identification of lynchpin urokinase plasminogen activator receptor protein interactions associated with epithelial cancer malignancy

Urokinase plasminogen activator (uPA) and its high affinity receptor (uPAR) play crucial proteolytic and non-proteolytic roles in cancer metastasis. In addition to promoting plasmin-mediated degradation of extracellular matrix barriers, cell surface engagement of uPA through uPAR binding results in the activation of a suite of diverse cellular signal transduction pathways. Because uPAR is bound to the plasma membrane through a glycosyl−phosphatidylinositol anchor, these signalling sequelae are thought to occur through the formation of multi-protein cell surface complexes involving uPAR. To further characterize uPAR-driven protein complexes, we co-immunoprecipitated uPAR from the human ovarian cancer cell line, OVCA 429, and employed sensitive proteomic methods to identify the uPAR-associated proteins. Using this strategy, we identified several known, as well as numerous novel, uPAR associating proteins, including the epithelial restricted integrin, αvβ6. Reverse immunoprecipitation using anti-β6 integrin subunit monoclonal antibodies confirmed the co-purification of this protein with uPAR. Inhibition of uPAR and/or β6 integrin subunit using neutralizing antibodies resulted in the inhibition of uPA-mediated ERK 1/2 phosphorylation and subsequent cell proliferation. These data suggest that the association of β6 integrin (and possibly other lynchpin cancer regulatory proteins) with uPAR may be crucial in co-transmitting uPA signals that induce cell proliferation. Our findings support the notion that uPAR behaves as a lynchpin in promoting tumorigenesis by forming functionally active multiprotein complexes.13 page(s