Abstract-Atherosclerosis is an inflammatory disease occurring preferentially in arterial regions exposed to disturbed flow conditions including oscillatory shear stress (OS). OS exposure induces endothelial expression of bone morphogenic protein 4 (BMP4), which in turn may activate intercellular adhesion molecule-1 (ICAM-1) expression and monocyte adhesion. OS is also known to induce monocyte adhesion by producing reactive oxygen species (ROS) from reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidases, raising the possibility that BMP4 may stimulate the inflammatory response by ROS-dependent mechanisms. Here we show that ROS scavengers blocked ICAM-1 expression and monocyte adhesion induced by BMP4 or OS in endothelial cells (ECs). Similar to OS, BMP4 stimulated H 2 O 2 and O 2 Ϫ production in ECs. Next, we used ECs obtained from p47phox Ϫ/Ϫ mice (MAE-p47 Ϫ/Ϫ ), which do not produce ROS in response to OS, to determine the role of NADPH oxidases. Similar to OS, BMP4 failed to induce monocyte adhesion in MAE-p47 Ϫ/Ϫ , but it was restored when the cells were transfected with p47 phox plasmid. Moreover, OS-induced O 2 Ϫ production was blocked by noggin (a BMP antagonist), suggesting a role for BMP. Furthermore, OS increased gp91phox (nox2) and nox1 mRNA levels while decreasing nox4. In contrast, BMP4 induced nox1 mRNA expression, whereas nox2 and nox4 were decreased or not affected, respectively. Also, OS-induced monocyte adhesion was blocked by knocking down nox1 with the small interfering RNA (siRNA). Finally, BMP4 siRNA inhibited OS-induced ROS production and monocyte adhesion. Together, these results suggest that BMP4 produced in ECs by OS stimulates ROS release from the nox1-dependent NADPH oxidase leading to inflammation, a critical early atherogenic step. Key words: BMP4 Ⅲ oscillatory shear Ⅲ reactive oxygen species Ⅲ monocyte adhesion Ⅲ endothelial cells Ⅲ NADPH oxidase V ascular endothelial cells (ECs) are constantly exposed to fluid shear stress, the frictional force generated by blood flow over the vascular endothelium. The importance of shear stress in vascular biology and pathophysiology has been highlighted by the focal development patterns of atherosclerosis in hemodynamically defined regions. For example, the regions of branched and curved arteries exposed to disturbed flow conditions including oscillatory shear stress (OS) correspond to "lesion-prone areas" that preferentially develop atherosclerosis. 1,2 In contrast, straight arteries exposed to steady, high levels of laminar shear stress (LS) are relatively well protected from atherosclerotic plaque development. 1,2 Atherosclerosis is an inflammatory disease preferentially occurring in lesion-prone areas. 2,3 The earliest measurable markers of atherogenesis include expression of inflammatory adhesion molecules such as E-selectin, vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1), and subsequent monocyte adhesion and recruitment into the lesion-prone areas. 2,4,5 Additional critical atherogen...

Objective-The similarities between valvular and vascular lesions suggest pathological initiation mediated through endothelium, but the role of hemodynamics in valvular endothelial biology is poorly understood. Methods and Results-Monolayers of porcine aortic endothelial cells (PAECs) or porcine aortic valve endothelial cells (PAVECs) were exposed to 20 dyne/cm 2 steady laminar shear stress for 48 hours, with static cultures serving as controls. Multiple microarray comparisons were made using RNA from sheared and control batches of both cell types. More than 400 genes were significantly differentially expressed in each comparison group. The resulting profiles were validated at the transcription and protein level and expression patterns confirmed in vivo by immunohistochemistry. PAVECs were found to be less intrinsically inflammatory than PAECs, but both cell types expressed similar antioxidant and antiinflammatory genes in response to shear stress. PAVECs expressed more genes associated with chondrogenesis, whereas PAECs expressed osteogenic genes, and shear stress had a protective effect against calcification. Conclusions-Transcriptional differences between PAVECs and PAECs highlight the valvular endothelial cell as a distinct organ system and suggest more attention needs to be given to valvular cells to further our understanding of similarities and differences between valvular and vascular pathology. Key Words: aortic valve Ⅲ shear stress Ⅲ inflammation Ⅲ calcification Ⅲ endothelial cell A ortic valve disease is associated with significant mortality and morbidity and is a strong risk factor for additional cardiovascular events. 1,2 Valvular degeneration is characterized by the development of stenosis or insufficiency, and by the time it is clinically manifested, it is usually only treatable by prosthetic valve replacement. 3 Explants of diseased valves reveal a wide spectrum of pathology, including sclerotic and calcific lesions, thrombus formations, bacterial vegetations, and fractured matrix fibers. 4,5 Aortic valve disease was originally thought to be the result of the continuous barrage of hemodynamic and mechanical forces over time, but recent evidence suggests a much more active biological progression involving inflammation, oxidation, angiogenesis, calcification, and osteogenesis. 6 -8 The vascular endothelium is a critical mediator of hemodynamic and humoral stimuli, and that endothelial inflammation and atherosclerosis occur preferentially at sites of disturbed or oscillatory flow. 9 Valvular endothelial dysfunction is also a hallmark of leaflet degeneration, and similarly characterized by the expression of proinflammatory adhesion receptors. 10,11 Interestingly, much of the aforementioned valvular pathology seems to occur preferentially on the aortic surface of the leaflet, which experiences a complex circulating flow that is different from the unidirectional flow on the ventricular side of the leaflet. This suggests that disturbed flow may play a causal role in the initiation of valvular pathology...

Sepsis is a deadly disease characterized by the inability to regulate the inflammatory–coagulation response in which the endothelium plays a key role. The cause of this perturbation remains poorly understood and has hampered the development of effective therapeutics. Matrix metalloproteases (MMPs) are involved in the host response to pathogens, but can also cause uncontrolled tissue damage and contribute to mortality. We found that human sepsis patients had markedly elevated plasma proMMP-1 and active MMP-1 levels, which correlated with death at 7 and 28 days after diagnosis. Likewise, septic mice had increased plasma levels of the MMP-1 ortholog, MMP-1a. We identified mouse MMP-1a as an agonist of protease-activated receptor-1 (PAR1) on endothelial cells. MMP-1a was released from endothelial cells in septic mice. Blockade of MMP-1 activity suppressed endothelial barrier disruption, disseminated intravascular coagulation (DIC), lung vascular permeability as well as the cytokine storm and improved survival, which was lost in PAR1-deficient mice. Infusion of human MMP-1 increased lung vascular permeability in normal wild-type mice but not in PAR1-deficient mice. These findings implicate MMP-1 as an important activator of PAR1 in sepsis and suggest that therapeutics that target MMP1-PAR1 may prove beneficial in the treatment of sepsis.

Ovarian cancer is a lethal gynecologic malignancy that may benefit from new therapies that block key paracrine pathways involved in tumor-stromal interactions and tumor vascularity. It was recently shown that matrix metalloprotease-1 (MMP1) activation of the G protein-coupled receptor protease-activated receptor-1 (PAR1) is an important stimulator of angiogenesis and metastasis in peritoneal mouse models of ovarian cancer. In the present study, we tested the hypothesis that MMP1-PAR1 promotes angiogenesis through its paracrine control of angiogenic chemokine receptors. We found that MMP1-PAR1 activation induces the secretion of several angiogenic factors from ovarian carcinoma cells, most prominently interleukin (IL)-8, growthregulated oncogene-α (GRO-α), and monocyte chemoattractant protein-1. The secreted IL-8 and GRO-α acts on endothelial CXCR1/2 receptors in a paracrine manner to cause robust endothelial cell proliferation, tube formation, and migration. A cell-penetrating pepducin, X1/2pal-i3, which targets the conserved third intracellular loop of both CXCR1 and CXCR2 receptors, significantly inhibited endothelial cell proliferation, tube formation, angiogenesis, and ovarian tumor growth in mice. Matrigel plugs mixed with MMP1-stimulated, OVCAR-4-conditioned media showed a dramatic 33-fold increase in blood vessel formation in mice. The X1/2pal-i3 pepducin completely inhibited MMP1-dependent angiogenesis compared with a negative control pepducin or vehicle. Conversely, a vascular endothelial growth factor-directed antibody, Avastin, suppressed angiogenesis in mice but, as expected, was unable to inhibit IL-8 and GRO-α-dependent endothelial tube formation in vitro. These studies identify a critical MMP1-PAR1-CXCR1/2 paracrine pathway that might be therapeutically targeted for ovarian cancer treatment. Cancer Res; 70(14); 5880-90. ©2010 AACR.

Protease-activated receptors (PARs) are G-proteincoupled receptors that are activated by proteolytic cleavage and generation of a tethered ligand. High PAR1 expression has been documented in a variety of invasive cancers of epithelial origin. In the present study, we investigated the contribution of the four PAR family members to motility of lung carcinomas and primary tumor samples from patients. We found that of the four PARs, only PAR1 expression was highly increased in the lung cancer cell lines. Primary lung cancer cells isolated from patient lung tumors migrated at a 10-to 40-fold higher rate than epithelial cells isolated from nonmalignant lung tissue. Cellpenetrating pepducin inhibitors were generated against the first (i1) and third (i3) intracellular loops of PAR1 and tested for their ability to inhibit PAR1-driven migration and extracellular regulated kinase (ERK)1/2 activity. The PAR1 pepducins showed significant inhibition of cell migration in both primary and established cell lines similar to silencing of PAR1 expression with short hairpin RNA (shRNA). Unlike i1 pepducins, the i3 loop pepducins were effective inhibitors of PAR1-mediated ERK activation and tumor growth. Comparable in efficacy with Bevacizumab, monotherapy with the PAR1 i3 loop pepducin P1pal-7 provided significant 75% inhibition of lung tumor growth in nude mice. We identify the PAR1-ERK1/2 pathway as a feasible target for therapy in lung cancer. Lung cancer is the leading cause of cancer deaths in the United States and worldwide, and is the second most common cancer overall. 1 The majority of patients eventually develop distant metastases, which leads to substantial morbidity and mortality. Currently available chemotherapeutic regimens for the treatment of non-small-cell lung cancer (NSCLC) include combinations of cisplatin or carboplatin, and etoposide, paclitaxel, docetaxel, gemcitabine, vinorelbin, and irinotecan. These regimens are generally not curative and may confer modest prolongation of life and symptomatic relief. 2,3 More recently, targeted therapies have become available for the treatment of lung cancer. These include small molecules and antibodies that target epidermal growth factor receptor and vascular endothelial growth factor receptor. However, the currently available molecular therapies still result in relatively modest prolongation of median and overall survival, pointing to the necessity for developing more effective treatment modalities for patients with advanced NSCLC.Emerging evidence has identified protease activated receptor-1 (PAR1) as a promising target to impact tumor progression, metastasis, and angiogenesis in a variety of cancers including breast, ovarian, melanoma, prostate, and colon cancer. 4 -7 However, the role of PAR1 and the other PAR family members in lung cancer is largely unexplored. To date, four different PARs have been identified: PAR1, PAR2, PAR3, and PAR4. 8,9 -13 PAR1 originally was discovered on platelets and serves as the prototype for this specialized class of proteolytically activ...

Objective-Fluid shear stress plays a role in angiogenesis. Laminar shear stress (LS) promotes endothelial cell (EC) quiescence, whereas oscillatory shear stress (OS) promotes EC turnover and dysfunction, which could lead to pathological angiogenesis. We hypothesized that LS inhibits EC migration and tubule formation, 2 functions important in angiogenesis, by inhibiting the secretion of proangiogenic factors. Methods and Results-Human umbilical vein ECs (HUVECs), human microvascular ECs (HMECs), or bovine aortic ECs (BAECs) were subjected to either LS (15 dyn/cm 2 ) or OS (Ϯ5 dyn/cm 2 ) for 24 hours and used in Matrigel tubule formation or scratch migration assays. Exposure of HUVECs, HMECs, but not BAECs, to LS inhibited tubule formation compared with OS. LS also inhibited migration of HUVECs and BAECs compared with OS. Angiopoietin-2 (Ang2), a known angiogenic protein, was found to be downregulated by LS both in cultured ECs and mouse aortas. Using Ang2 siRNA, Ang2 knockdown blocked OS-mediated migration and tubule formation and the LS-inhibited tubule formation was partially rescued by recombinant Ang2. Conclusions-Our data suggests that Ang2 produced by OS in ECs plays a critical role in migration and tubule formation, and may play an important role in diseases with disturbed flow and angiogenesis. (Arterioscler Thromb Vasc Biol. 2007;27:2150-2156.)Key Words: shear stress Ⅲ endothelial cells Ⅲ angiogenesis Ⅲ angiopoietin-2 F luid shear stress, the dragging force created by flow through blood vessels, is sensed by the endothelium and plays an important role in normal physiological responses as well as disease pathologies. In particular, shear stress is thought to play a role in angiogenesis, or the formation of new blood vessels from preexisting blood vessels. 1-3 At the cellular and molecular level, unidirectional laminar shear stress (LS) is thought to promote endothelial cell (EC) quiescence; laminar sheared ECs are antiproliferative, antiapoptotic, and antithrombotic. 4 However, oscillatory shear stress (OS), a type of disturbed shear stress implicated in diseases such as atherosclerosis, is thought to promote EC dysfunction; oscillatory sheared ECs are proproliferative, promigratory, prothrombotic, and secrete growth factors that stimulate smooth muscle cell proliferation and migration. 4 The secretion of growth factors from dysfunctional ECs exposed to OS could also play a role in blood vessel remodeling and angiogenesis.There are several diseases associated with both angiogenesis and disturbed flow, such as atherosclerosis, aortic valve calcification, and arterial occlusion. In atherosclerosis, atherosclerotic plaques preferentially occur in areas of the arterial system exposed to disturbed flow and angiogenesis in the plaque is thought to promote the progression of atherosclerosis. 5,6 Disturbed flow is found in the aortic valve sinus and angiogenesis in the valve leaflet is associated with calcification. 7,8 In arterial occlusion, disturbed flow is found in the postocclusive site and angiogenesis is...

Most ovarian cancer patients are diagnosed late in progression and often experience tumor recurrence and relapses due to drug resistance. Surface expression of matrix metalloprotease MMP-14 on ovarian cancer cells stimulates a tumor-stromal signaling pathway that promotes angiogenesis and tumor growth. In a cohort of 92 patients, we found that MMP-14 was increased in the serum of women with malignant ovarian tumors. Therefore, we investigated the preclinical efficacy of a MMP-14 monoclonal antibody that could inhibit the migratory and invasive properties of aggressive ovarian cancer cells in vitro. MMP-14 antibody disrupted ovarian tumor-stromal communication and was equivalent to Avastin in suppressing blood vessel growth in mice harboring matrigel plugs. These effects on angiogenesis correlated with down regulation of several important angiogenic factors. Further, mice with ovarian cancer tumors treated with anti-MMP-14 monotherapy showed a marked and sustained regression in tumor growth with decreased angiogenesis compared to IgG treated controls. In a model of advanced peritoneal ovarian cancer, MMP-14-dependent invasion and metastasis was effectively inhibited by intraperitoneal administration of monoclonal MMP-14 antibody. Together, these studies provide a preclinical proof-of-concept for MMP-14 targeting as an adjuvant treatment strategy for advanced ovarian cancer.

G protein-coupled receptors (GPCR) are a superfamily of receptors that are vital in a wide array of physiological processes. Modulation of GPCR signaling has been an intensive area of therapeutic study, mainly due to the diverse pathophysiological significance of GPCRs. Pepducins are cell-penetrating lipidated peptides designed to target the intracellular loops of the GPCR of interest. Pepducins can function as agonists or antagonists of their cognate receptor, making them highly useful compounds for the study of GPCR signaling. Pepducins have been used to control platelet-dependent hemostasis and thrombosis, tumor growth, invasion, and angiogenesis, as well as to improve sepsis outcomes in mice. Pepducins have been successfully designed against a wide variety of GPCRs including the protease-activated receptors (PAR1, 2, 4), the chemokine receptors (CXCR1, 2, 4), the sphingosine-1-phosphate receptor (S1P3), the adrenergic receptor (ADRA1B), and have the potential to help reveal the functions of intractable GPCRs. Pharmacokinetic, pharmacodynamic, and biodistribution studies have showed that pepducins are widely distributed throughout the body except the brain and possess appropriate drug-like properties for use in vivo. Here, we discuss the delivery, pharmacology, and biodistribution of pepducins, as well as the effects of pepducins in models of inflammation, cardiovascular disease, cancer, and angiogenesis.