Changes In Vascular Permeability Research Articles

The effect of AMG 706, a novel multi-kinase inhibitor, on vascular permeability and blood flow as assessed by dynamic contrast enhanced magnetic resonance imaging in an in vivo preclinical tumor model

3134 Background: AMG 706 is a potent, oral, multi-kinase inhibitor with anti-angiogenic and anti-tumor activity achieved by selectively targeting all known VEGF, PDGF, Kit and Ret receptors. In previous experiments, AMG 706 treatment resulted in regression of large, established A431 epidermoid carcinoma tumors within seven days of the initiation of treatment. The present experiments were carried out to relate the therapeutic effect to changes in vascular permeability and blood flow, with the intent of identifying a pharmacodynamic marker for use in clinical trials. Methods: After an initial dynamic contrast enhanced magnetic resonance imaging (DCE-MRI) baseline scan, mice bearing A431 xenografts (300 mm3) were dosed with either vehicle or AMG 706 at dose levels ranging between 10 and 75 mg/kg. DCE-MRI was repeated after 24, 48 or 96 hrs of treatment. All MRI studies were performed at 4.7 Tesla (Bruker Biospin, Billerica, MA). During the DCE-MRI scans, 0.2 mmol/kg gadopentetate dimeglumine was injected intravenously via a tail vein catheter. Results: DCE-MRI analysis identified a statistically significant (p = 0.0235) decrease in contrast agent uptake (AUC relative to baseline), occurring in a dose dependent fashion in the interior region of the tumor after only 24 hrs of treatment with AMG 706. The effect seen at 24 hrs is consistent with the previous observation that tumor endothelial cell apoptosis occurs within 12 hrs of initiation of treatment, and indicates a statistically significant treatment related reduction in tumor blood flow and vascular permeability in the A431 tumor model. Conclusions: Oral administration of AMG 706 produced a statistically significant reduction in vascular blood flow and/or permeability in the A431 human tumor xenograft model as early as 24 hrs of treatment. These results are consistent with previous observations indicating the destruction of tumor blood vessels as the mechanism of action of AMG 706, and support the use of DCE-MRI to obtain pharmacodynamic information in clinical trials of AMG 706. Author Disclosure Employment or Leadership Consultant or Advisory Role Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration Amgen Amgen Amgen

Anthrax Lethal Toxin Induces Endothelial Barrier Dysfunction

Hemorrhage and pleural effusion are prominent pathological features of systemic anthrax infection. We examined the effect of anthrax lethal toxin (LT), a major virulence factor of Bacillus anthracis, on the barrier function of primary human lung microvascular endothelial cells. We also examined the distribution patterns of cytoskeletal actin and vascular endothelial-cadherin (VE-cadherin), both of which are involved in barrier function regulation. Endothelial monolayers cultured on porous membrane inserts were treated with the LT components lethal factor (LF) and protective antigen (PA) individually, or in combination. LT induced a concentration- and time-dependent decrease in transendothelial electrical resistance that correlated with increased permeability to fluorescently labeled albumin. LT also produced a marked increase in central actin stress fibers and significantly altered VE-cadherin distribution as revealed by immunofluorescence microscopy and cell surface enzyme-linked immunosorbent assay. Treatment with LF, PA, or the combination of an inactive LF mutant and PA did not alter barrier function or the distribution of actin or VE-cadherin. LT-induced barrier dysfunction was not dependent on endothelial apoptosis or necrosis. The present findings support a possible role for LT-induced barrier dysfunction in the vascular permeability changes accompanying systemic anthrax infection.

Hepatic arterial infusion (HAI) therapy in patients with unresectable primary liver cancer: Use of dynamic contrast enhanced MRI to evaluate response

4129 Background: This study reports preliminary results of the use of HAI of Floxuridine (FUDR) and Dexamethasone (Dex) in patients (pts) with unresectable cholangiocarcinoma (CC) or hepatocellular cancer (HCC), and investigates the use of dynamic contrast-enhanced MRI (DCE-MRI) to evaluate changes in tumor perfusion, and to correlate these findings with radiographic tumor response. Methods: Seventeen pts (15 CC, 2 HCC) deemed unresectable by experienced hepatobiliary surgeons were treated with HAI FUDR/Dex via arterial pump by continuous infusion over 14 days on a 4-week cycle. Imaging: MRI imaging was performed on a conventional 1.5 Tesla MRI scanner. DCE-MRI images were acquired with a temporal resolution of 1 second per image. Analysis was performed with a two compartment model, the slope of the initial contrast uptake, as well as transfer between lesion and plasma compartments were calculated. Results: Of 17 pts, 14 are presently evaluable, while 3 are too early to assess for response. One of 2 pts with HCC responded; 5 of 12 pts (42%) with CC had partial response, and 6 (50%) had stable disease. Median duration of response is 11.9 months (range 4.1 - 14.7). Median time to progression for all pts is 7.2 months. Seven have progressed: 4 (liver), 2 (lung), 1 (bone). Treatment has been well-tolerated: Gr 3 SGOT elevation was seen in 5 pts (29%), and Gr 3 alkaline phosphatase in 4 (23%). No pt had bilirubin elevation, diarrhea or neutropenia. Comparison of lesion size on DCE-MRI and conventional MRI showed that post-treatment changes in tumor perfusion (slope measurements) observed at 2 months predict treatment response earlier than bi-dimensional tumor measurements. Patients with rapid uptake of contrast (slope > 300% increase/min) all showed tumor response. Conclusions: Presently, HAI therapy with FUDR + Dex demonstrates a 43% response rate with an excellent toxicity profile. Changes in tumor perfusion and vascular permeability may predict treatment response earlier and correlate with probability of response in pts with CC or HCC. Author Disclosure Employment or Leadership Consultant or Advisory Role Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration Johnson & Johnson Johnson & Johnson

Inhibition of vessel permeability by TNP-470 and its polymer conjugate, caplostatin

Angiogenesis inhibitors, such as TNP-470 and the nontoxic HPMA copolymer-TNP-470 (caplostatin), are emerging as a class of anticancer drugs. We report that TNP-470 and caplostatin inhibit vascular hyperpermeability of tumor blood vessels as well as that induced in mouse skin by different mediators. Treatment with TNP-470 or angiostatin for 3 days was sufficient to reduce permeability of tumor blood vessels, delayed-type hypersensitivity, and pulmonary edema induced by IL-2. TNP-470 also inhibited VPF/VEGF-induced phosphorylation of VEGFR-2, calcium influx, and RhoA activation in endothelial cells. These results identify an activity of TNP-470, that of inhibiting vessel hyperpermeability. This activity likely contributes to TNP-470's antiangiogenic effect and suggests that caplostatin can be used in the treatment of cancer and inflammation.

The Thioredoxin-1 Inhibitor 1-Methylpropyl 2-Imidazolyl Disulfide (PX-12) Decreases Vascular Permeability in Tumor Xenografts Monitored by Dynamic Contrast Enhanced Magnetic Resonance Imaging

Abstract Purpose: The purpose of this study was to use dynamic contrast enhanced magnetic resonance imaging (DCE-MRI) to measure changes in tumor xenograft permeability produced by the antitumor thioredoxin-1 (Trx-1) inhibitor 1-methylpropyl 2-imidazolyl disulfide (PX-12) and to assess the relationship to Trx-1 and vascular endothelial growth factor (VEGF) levels. Experimental Design: DCE-MRI was used to monitor the dynamics of gadolinium-diethylenetriaminepentaacetic acid coupled bovine serum albumin as a macromolecular contrast reagent to measure hemodynamic changes in HT-29 human colon xenografts in immunodeficient mice treated with PX-12. Blood vessel permeability was estimated from the slope of the enhancement curves, and tumor vascular volume fraction from the ordinate. Tumor Trx-1 and VEGF was also measured. Results: PX-12 caused a rapid 63% decrease in the average tumor blood vessel permeability within 2 hours of administration. The decrease lasted 24 hours and had returned to pretreatment values by 48 hours. The changes in vascular permeability were not accompanied by alterations in average tumor vascular volume fraction. There was a decrease in tumor and tumor-derived VEGF in plasma at 24 hours after treatment with PX-12, but not at earlier time points. However, tumor redox active Trx-1 showed a rapid decline within 2 hours following PX-12 administration that was maintained for 24 hours. Conclusion: The rapid decrease in tumor vascular permeability caused by PX-12 administration coincided with a decrease in tumor redox active Trx-1 and preceded a decrease in VEGF. DCE-MRI responses to PX-12 in patients of Trx-1 inhibition at early time points and decreased VEGF at later times, may be useful to follow tumor response and even therapeutic benefit.

A Mouse Model for the Study of Vascular Permeability Changes Induced by Arsenic

The primary objective of our present research was to develop an animal model for the investigation of arsenic-induced vasculopathy. Epidemiological evidence indicated that, aside from cancer, cardiovascular-related diseases were probably the most prominent health concerns in arseniasis areas. Although there were many investigations on the effect of arsenic on endothelial cells, most of these studies were conducted under in vitro conditions. A good animal model for studying the effects of arsenic on vascular integrity in vivo is very much needed. We have previously developed a rat model that could be used to demonstrate vascular changes induced by arsenic in vivo. In the present report, we are introducing a new model that is even more sensitive, economical, and effective than our original rat model. Taking advantage of the characteristics of mouse ears (thin, delicate, and with easily visible blood vessels), we demonstrated the pattern and extent of vascular leakage induced by arsenic clearly, quantitatively and convincingly. With this model, we demonstrated a time-dependent increase in vascular permeability induced by arsenic (a fourfold increase in vascular leakage was observed between 10–60 minutes). With this model, we were also able to demonstrate that small caliber vessels were more vulnerable than vessels of larger caliber. Thus, this animal model can provide dynamic information (from toxic effects, pathology, and functional alterations to cellular/molecular mechanisms) that cell culture techniques are unable to provide. The introduction of this model hopefully will stimulate and inspire many other new in vivo investigations on vasculopathy induced by arsenic or other chemicals in the future.

Antipruritic and Antierythema Effects of Ascomycete Bulgaria inquinans Extract in ICR Mice

The effect of ethanol extract obtained from Bulgaria inquinans on the scratching behavior and vascular permeability changes induced by compound 48/80, histamine and serotonin in ICR mice was studied. The extract dose-dependently inhibited scratching behavior induced by compound 48/80 and serotonin. A significant inhibition was observed at doses of 300 and 600 mg/kg when Bulgaria inquinans extract was administered orally. However, no inhibitory effect was observed on the histamine-induced scratching behavior by the extract, even at a dose of 600 mg/kg. In addition, it also inhibited the increase in the vascular permeability induced by compound 48/80 and serotonin at doses of 300 and 600 mg/kg; however, it failed to inhibit the increased vascular permeability induced by histamine, even at a dose of 600 mg/kg. Bulgaria inquinans extract showed a potent inhibitory effect on histamine release induced by compound 48/80. These results suggest that Bulgaria inquinans extract is effective in cutaneous pruritus and erythema, which were probably mediated by inhibiting the release of histamine from mast cells and antagonizing the effect on serotonin.

Enhanced dermal and retinal vascular permeability in streptozotocin-induced type 1 diabetes in Wistar rats: blockade with a selective bradykinin B 1 receptor antagonist

The vascular complications associated with type 1 diabetes are to some extent related to the dysfunction of the endothelium leading to an increased vascular permeability and plasma extravasation in the surrounding tissues. The various micro- and macro-vascular complications of diabetes develop over time, leading to nephropathy, retinopathy and neuropathy and cardiomyopathy. In the present study, the effect of a novel selective bradykinin B 1 receptor (BKB 1-R) antagonist, R-954, was investigated on the changes of vascular permeability in the skin and retina of streptozotocin (STZ)-induced type 1 diabetic rats. Plasma extravasation increased in the skin and retina of STZ-diabetic rats after 1 week and persisted over 4 weeks following STZ injection. Acute treatment with R-954 (2 mg/kg, bolus s.c.) highly reduced the elevated vascular permeability in both 1- and 4-week STZ-diabetic rats. These results showed that the inducible BKB 1-R subtype modulates the vascular permeability of the skin and retina of type 1 diabetic rats and suggests that BKB 1-R antagonists could have a beneficial role in diabetic neuropathy and retinopathy.

Chylomicron-bound LPS inhibits tumor necrosis factor-induced capillary leak in vivo

TNF plays a central role in the host acute inflammatory response during sepsis. A potent mediator of inflammation, this cytokine has been shown to increase microvascular permeability through a direct effect on endothelial cells. Previously, we have demonstrated that chylomicron (CM)-bound LPS inhibits the hepatocellular response to TNF-induced stimulation. Therefore, we hypothesized that CM-LPS can also protect endothelial cells from the deleterious effects of TNF. Sprague-Dawley rats were injected with saline (control), CM (375 mg/kg BW), LPS (19 μg/kg BW), or combined CM-LPS complexes, 4 h prior to TNF exposure. We then examined the effect of TNF (1 μg/mL) applied intraluminally on microvascular permeability (hydraulic conductivity) in post-capillary venules using the Landis technique. As expected, TNF stimulation increased vascular permeability approximately 3-fold as compared to baseline (Fig. 1). CM alone partially inhibited the TNF-induced increased permeability. However, pretreatment with CM-LPS completely abolished the TNF-induced changes in vascular permeability. Conversely, the animals injected with LPS alone yielded an exaggerated response to TNF. CM-LPS inhibited TNF-induced increases in vascular permeability in vivo. Consistent with our previous observations in hepatocytes, our data suggest that lipoprotein-bound LPS can similarly induce cytokine tolerance in vascular endothelial cells, yielding further support for the development of a lipid-based therapy for gram negative sepsis.

A phase I/II trial of single-agent PTK 787/ZK 222584 (PTK/ZK), a novel, oral angiogenesis inhibitor, in patients with recurrent glioblastoma multiforme (GBM)

1512 Background: VEGF-mediated signaling pathways may be critical to GBM neovascularization, and inhibition of VEGF pathways may be an important therapeutic target in GBM. PTK/ZK is a novel, oral angiogenesis inhibitor that potently inhibits all known vascular endothelial growth factor receptors (VEGFRs), and therefore blocks signaling by VEGF-A, -B, -C, and -D. Methods: PTK/ZK was administered once daily as oral monotherapy starting at 150 mg/day and escalating to 500, 1,000, 1,500, or 2,000 mg/day, with dose expansion at the 1,200- and 1,500-mg/day dose levels. The primary objective was to determine the safety and tolerability of continuous oral PTK/ZK in patients with recurrent GBM. A secondary endpoint was response to treatment (defined as decreases in tumor blood supply by dynamic contrast-enhanced [DCE] and dynamic susceptibility-change [DSC] MRI) and tumor response. Results: To date, 55 patients have been treated at these dose levels: 150 mg/day (n = 3); 500 mg/day (n = 6); 1,000 mg/day (n = 6); 1,200 mg/day (n = 20); 1,500 mg/day (n = 14); and 2,000 mg/day (n = 6). Median age was 54 years (range, 21–73 years), and median prior number of therapies was 4 (range, 1–12 therapies). Dose-limiting toxicities included liver enzyme elevation and deep-vein thrombosis (in 1/6 patients at 1,000 mg/day), insomnia or cerebral edema (in 2/14 patients at 1,500 mg/day), and fatigue or nausea/vomiting (in 2/6 patients at 2,000 mg/day). Among 47 evaluable patients, best responses were 2 (4%) partial responses, 31 (56%) stable disease, and 14 (25%) disease progression. Median duration of stable disease was 12.1 weeks (95% CI = 9.9, 15.1 weeks). DCE- and DSC-MRI scanning showed decreases in vascular permeability and cerebral blood volume, respectively, at days 2 and 30 of treatment; the decreases in both parameters at day 30 appeared dose dependent. Further assessment by blinded reviewers is ongoing. Conclusions: PTK/ZK monotherapy at 1,200 and 1,500 mg/day was well tolerated in patients with recurrent GBM, demonstrated changes in vascular permeability and blood volume, and stabilized disease in the majority of patients. Author Disclosure Employment or Leadership Consultant or Advisory Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration Novartis Pharmaceuticals Corp.; Schering AG Imclone; Novartis; Schering-Plough Imclone; Novartis; Regeneron; Schering-Plough; Schering AG Novartis

Direct stimulation of adult neural stem cells in vitro and neurogenesis in vivo by vascular endothelial growth factor.

Hypoxia as well as global and focal ischemia are strong activators of neurogenesis in the adult mammalian central nervous system. Here we show that the hypoxia-inducible vascular endothelial growth factor (VEGF) and its receptor VEGFR-2/Flk-1 are expressed in clonally-derived adult rat neural stem cells in vitro. VEGF stimulated the expansion of neural stem cells whereas blockade of VEGFR-2/Flk-1-kinase activity reduced neural stem cell expansion. VEGF was also infused into the lateral ventricle to study changes in neurogenesis in the ventricle wall, olfactory bulb and hippocampus. Using a low dose (2.4 ng/d) to avoid endothelial proliferation and changes in vascular permeability, VEGF stimulated adult neurogenesis in vivo. After VEGF infusion, we observed reduced apoptosis but unaltered proliferation suggesting a survival promoting effect of VEGF in neural progenitor cells. Strong expression of VEGFR-2/Flk-1 was detected in the ventricle wall adjacent to the choroid plexus, a site of significant VEGF production, which suggests a paracrine function of endogenous VEGF on neural stem cells in vivo. We propose that VEGF acts as a trophic factor for neural stem cells in vitro and for sustained neurogenesis in the adult nervous system. These findings may have implications for the pathogenesis and therapy of neurodegenerative diseases.

Prostaglandin E2 Is a Product of Induced Prostaglandin-endoperoxide Synthase 2 and Microsomal-type Prostaglandin E Synthase at the Implantation Site of the Hamster

Certain uterine prostaglandins (PGs) are elevated at implantation sites and are needed to trigger the events of blastocyst implantation that include blastocyst-uterine attachment and stromal decidualization with vascular permeability changes. Several decades of investigations showed that treatment with PG synthesis inhibitors, prior to or during the time of implantation, resulted in either complete inhibition or a delay in implantation or reduction in the number of implantation sites with diminished decidual tissue. Consistent with these findings, we observed that whereas a selective PG endoperoxide synthase (Ptgs) 1 inhibitor SC-560 failed to inhibit implantation, a selective Ptgs2 inhibitor SC-236 showed significantly reduced number and size of implantation sites in progesterone-treated ovariectomized pregnant hamsters. It is known that Ptgs2 expression and Ptgs2-derived prostacyclin (PGI2) synthesis at implantation sites are needed for implantation in the mouse (a rodent that needs ovarian estrogen for implantation). However, it is unknown which Ptgs and PG synthases produce which PGs at implantation sites of the hamster (a rodent that does not need ovarian estrogen for implantation). Here we demonstrate that as blastocyst implantation proceeds, a reduction in Ptgs1 expression from uterine luminal epithelial cells and a gradual induction in Ptgs2 expression exclusively in luminal epithelial and adjacent decidual cells occurred at implantation sites of hamsters. Results also reveal that PGE2, but not PGI2, is the major PG at implantation sites where Ptgs2 and microsomal type PGE synthases but not PGI synthases are co-expressed. This elevated uterine PGE2 at implantation sites may serve to initiate or amplify physiological signals required for specific aspects of the implantation process in hamsters.

β-chemokine function in experimental lung ischemia-reperfusion injury

Background Although chemokines are functionally important in models of ischemia-reperfusion injury, little is known about their role in lung ischemia-reperfusion injury (LIRI). This study examined the role of the β-chemokines, macrophage inflammatory protein (MIP)-1α, monocyte chemoattractant protein (MCP)-1, and regulated upon activation normal T cells expressed and secreted (RANTES) in LIRI. Methods Left lungs of Long-Evans rats underwent normothermic ischemia for 90 minutes and reperfusion for up to 4 hours. Treated animals received anti–MIP-1α, anti–MCP-1, or anti-RANTES antibodies before reperfusion. Changes in lung vascular permeability were measured with iodine 125–labeled bovine serum albumin. Neutrophil accumulation in the lung parenchyma was determined by myeloperoxidase activity, and bronchoalveolar lavage was performed to measure leukocyte cell counts. Western blots, Northern blots, and ribonuclease protection assays assessed β-chemokine messenger RNA and protein levels. Results Animals receiving anti–MIP-1α demonstrated reduced vascular permeability compared with controls ( p < 0.001). Attenuation of permeability was less dramatic in animals treated with anti–MCP-1 and anti-RANTES antibody, which demonstrated permeability decreases of 15% and 16%, respectively ( p < 0.02). Lung neutrophil accumulation was reduced in animals receiving anti–MIP-1α antibody ( p < 0.005) but was unchanged in animals receiving either anti–MCP-1 or anti-RANTES. Bronchoalveolar lavage leukocyte content was also reduced by treatment with anti–MIP-1α ( p < 0.003) and was unchanged in anti–MCP-1–treated and anti-RANTES–treated animals. MIP-1α treatment decreased tumor necrosis factor-α messenger RNA in injured left lungs. Conclusions MIP-1α is functionally significant in the development of LIRI. It likely exerts its effects in part by mediating the expression of proinflammatory and antiinflammatory cytokines and influencing tissue neutrophil recruitment. MCP-1 and RANTES seem to play relatively minor roles in the development of direct LIRI.

Physiological mechanisms underlying heat-induced radiosensitization

The objective of this review is to evaluate hyperthermia related changes in tumor physiologic parameters and their relevance for tumor radiosensitization with particular emphases on tumor oxygenation. Elevation of temperature above the physiological level causes changes in blood flow, vascular permeability, metabolism, and tumor oxygenation. These changes in addition to the cellular effects such as direct cytotoxicity, inhibition of potentially lethal damage and sublethal damage repair, have an important influence on the efficacy of radiotherpay. There is now clear evidence that in a variety of rodent and canine, as well as human tumors, the changes in tumor oxygenation status caused by hyperthermia are temperature dependent and this relationship may greatly influence the response of tumors to thermo-radiotherapy. The improvement of tumor oxygenation after mild hyperthermia, which often lasts for as long as 24–48 h after heating, may increase the likelihood of a positive response of tumors to radiation therapy. Furthermore, the activity of some chemotherapy drugs is also oxygen dependent, therefore, the heat-induced increase in tumor oxygenation may significantly increase the effectiveness of thermoradiotherapy in combination with certain chemotherapy drugs. Further investigations remain to be conducted to obtain clearer insights into the relationship between thermal parameters, oxygenation and response of human tumors to hyperthermia in combination with radiotherapy and/or chemotherapy.

Radiation-induced edema is dependent on cyclooxygenase 2 activity in mouse brain.

Cerebrovascular dysfunction, characterized by compromise of the blood-brain barrier and formation of cerebral edema, is common during the acute period after brain irradiation and may contribute to delayed pathology (e.g. vascular collapse, white matter necrosis) that leads to functional deficits. Another response of normal brain tissue to radiation is the induction of inflammatory markers, such as cytokine expression and glial activation. In particular, radiation-induced neuroinflammation is associated with an elevation in cyclooxygenase 2 (COX2), one of two isoforms of the obligate enzyme in prostanoid synthesis and the principal target of non-steroid anti-inflammatory drugs. Since prostanoids serve as autocrine and paracrine mediators in numerous physiological and pathological processes, including vasoregulation, we investigated COX2 protein expression and COX2-mediated prostanoid production in radiation-induced cerebral edema in male C57/BL6 mice. We found that radiation induces COX2 protein that is accompanied by specific increases in prostaglandin E(2) and thromboxane A(2) within 4 and 24 h after brain irradiation. Furthermore, we showed that treatment with NS-398, a selective COX2 inhibitor, attenuated prostanoid induction and edema formation. These results suggest that radiation-induced changes in vascular permeability are dependent on COX2 activity, implicating this enzyme and its products as targets for potential therapeutic treatment/protection from the effects of radiation on normal brain tissue.

Do the signalling proteins for angiogenesis exist as a modular complex? The case for the angosome

The vasculature remains quiescent during much of adult life, but new blood vessels can be rapidly produced when required by a process referred to as angiogenesis. Angiogenesis involves a complex series of events including the proliferation, migration, differentiation and apoptosis of capillary endothelial cells, as well as changes in vascular permeability. This hypothesis argues that in the quiescent vasculature the many factors that regulate angiogenesis are normally held together as part of an inactive modular unit, and that when angiogenesis is stimulated the modular unit dissociates thus enabling angiogenic regulators to become active. I have termed this modular unit the “angosome”. It is proposed that the angosome is present in the caveolae of capillary endothelial cells. Caveolae are flask-shaped invaginations in the plasmalemma that compartmentalise signalling molecules. Endothelial cells are particularly rich in caveolae. Many of the structural and functional aspects of caveolae are controlled by the protein caveolin, one form of which, caveolin-1, interacts directly or indirectly with most of the regulatory molecules involved in angiogenesis. Caveolin-1 forms oligomers of 14–16 sub-units and I propose that oligomers of caveolin-1 form the scaffold that holds together the angosome. There is evidence that caveolin-1 is up-regulated in the differentiated, quiescent vasculature and down-regulated in proliferating endothelial cells. Since the presence of caveolin-1 can inhibit pro-angiogenic factors, it may act as a “master-switch” co-ordinating events during angiogenesis. Thus when the vasculature is quiescent the angosome may hold angiogenic factors in an inactive state and when angiogenesis is required, the angosome must disassociate to enable angiogenic factors to become active.

Vascular permeability alterations induced by arsenic.

The impact of arsenic on the integrity of blood vessels in vivo via in situ exposure (local injection) of arsenic was investigated. Vascular permeability changes were evaluated by means of the Evans blue assay and the India ink tracer techniques. Rats were intravenously injected with Evans blue followed by intradermal injections of various doses of sodium arsenite on the back skins of the animals. Evans blue at different time points was extracted and assayed as indices of vascular leakage. Skin at various time point injection sites was sampled for arsenic measurement via graphite furnace atomic absorption spectroscopy. Our time course study with Evans blue technique demonstrated a biphasic pattern of vascular permeability change: an early phase of permeability reduction and a later phase of permeability promotion at all dose levels tested. The India ink tracer technique also demonstrated a time-correlated increase in vascular labelling in the tissues examined, signifying an increase in vascular leakage with time. Moreover, we found that despite an early increase in tissue arsenic content at time of injection, tissue arsenic declined rapidly and returned to near control levels after 30-60 min. Thus, an inverse correlation between tissue arsenic content and the extent of vascular permeability was apparent. This study provides the first demonstration that in situ exposure to arsenic will produce vascular dysfunction (vascular leakage) in vivo.

Role of polymorphonuclear leukocytes, nitric oxide synthase, and cyclooxygenase in vascular permeability changes induced by C5a agonist peptides

Tumor responses to radioimmunotherapy combined with peptide agonists of human C5a anaphylatoxin such as GCGYSFKPMPLaR (C5aAP) are two- to four-fold better, depending on the dose of C5aAP, than responses to radioimmunotherapy alone. The enhanced tumor vascular permeability (VP) is the key factor responsible for this improvement. These studies were designed to identify the sequence of events leading to the improved extravasation of immunoglobulin in response to C5aAP. The VP changes were measured in mice after administration of C5aAP alongside of various mediators. The depletion of circulating polymorphonuclear neutrophils (PMN) in mice abolished the C5aAP-induced VP increase. Blocking of P-selectin also returned VP to its basal levels after the C5aAP treatment, indicating that C5aAP-induced VP changes are initiated by interactions of C5aAP with PMNs. Aminoguanidine, an inducible nitric oxide synthase (NOS) inhibitor, given before C5aAP returned VP to control levels. N(omega)-nitro-L-arginine methyl ester, a nonselective NOS inhibitor, had a marginal effect on the activity of C5aAP. Indomethacin, a nonselective cyclooxygenase inhibitor, suppressed C5aAP-induced increases in VP, whereas N-(2-cyclohexyloxy-4-nitrophenyl)-methanesulfonamide, a selective cyclooxygenase-2 inhibitor, was active only at high doses. While C5aAP given i.p. did not alter tumor uptake of (125)I-B72.3, the i.v. administration resulted in approximately 40% increase, confirming the prerequisite interaction of C5aAP with PMNs. The sequence leading to the increased VP appears to be initiated by the interaction of C5aAP with C5a receptor expressed on PMNs followed by binding to endothelial cells of blood vessels. The interaction with P-selectin is responsible for the initiation of the nitric oxide cascade as evidenced by inducible NOS activation. Additionally, prostaglandins are required for expression of the full magnitude of the C5aAP activities.

Erythropoietin Protects against Necrotizing Enterocolitis of Newborn Rats by the Inhibiting Nitric Oxide Formation

Background: Necrotizing enterocolitis (NEC) is an important neonatal disease with a high mortality rate; erythropoietin (Epo) is a hematopoietic growth factor. Functional Epo receptors are in the fetal and postnatal small bowel and their ligands are available for binding. Excessive nitric oxide (NO) production by an isoform of NO synthase inducible by inflammatory stimuli leads to changes in vascular permeability and tissue injury. The aim of this study was to investigate NO formation in an experimental model of NEC and the possible role of NO in the protection Epo provides against NEC. Methods: Twenty-four Wistar albino rat pups were divided into three groups: group 1 = control; group 2 = hypoxia-reoxygenation and saline; group 3 = hypoxia-reoxygenation and recombinant human EPO (rhEpo) pretreatment. rhEpo was given 750 U/kg/week by intraperitoneal injection 3 times a week for 2 weeks. On the 15th day, hypoxia was induced by placing the pups in a 100% CO₂ chamber for 5 min. After the hypoxia period the pups were reoxygenated for 10 min with 100% O₂ and returned to their mothers. All pups were killed 4 h after the hypoxia-reoxygenation period was over. The abdomen was opened and representative samples of injured areas were taken for histopathologic examination. Then nitrite levels were determined in the intestine by Griess Reagent. Results: On histopathological examination, injury scores in group-2 animals were found to be significantly higher than in group-3 animals (p = 0.001). Significantly increased intestinal nitrite levels were found in group-2 rats compared to the rats of groups 1 and 3 (p = 0.001 and p = 0.001, respectively). There was a positive correlation between the histological findings and the intestinal nitrite levels in group-2 and -3 animals (r = 0.94, p = 0.001; r = 0.99, p = 0.001, respectively). Conclusion: The present study demonstrates that the Epo-pretreated group had decreased levels of NO and limited mucosal necrosis in intestinal tissue samples. We believe that these results deserve further experimental studies in order to elucidate the possible effector mechanisms involved in the inhibitory relationship between Epo, NO and NEC.

Pathophysiological significance of peroxidative stress, neuronal damage, and membrane permeability in acute mountain sickness.

Free radical-mediated changes in vascular permeability and subsequent inflammatory response may be a contributory pathogenetic cofactor responsible for the development of neurological sequelae associated with acute mountain sickness (AMS). To investigate this, 49 subjects were examined at sea level and serially after rapid ascent to 4,559 m. Although the venous concentration of total creatine phosphokinase activity was measured in all subjects, a complementary examination of lipid peroxidation (F(2)-isoprostanes), inflammatory (TNF-alpha, IL-1beta, IL-2, IL-6, IL-8, C-reactive protein), and cerebrovascular tissue damage (neuron-specific enolase) biomarkers was confined to a subcohort of 24 subjects. A selective increase (P < 0.05) in total creatine phosphokinase was observed in subjects diagnosed with AMS at high altitude (n = 25) compared with apparently healthy controls (n = 24). However, despite a marked increase in IL-6 and C-reactive protein attributable primarily to subjects developing high-altitude pulmonary edema, subcohort analyses demonstrated no selective differences in F(2)-isoprostanes, neuron-specific enolase, or remaining proinflammatory cytokines due to AMS (n = 14). The present findings are the first to demonstrate that free radical-mediated neuronal damage of sufficient degree to be detected in the peripheral circulation does not occur and is, therefore, unlikely to be an important, initiating event that is critical for the development of AMS. The pathophysiological significance of increased sarcolemmal membrane permeability and inflammatory response, either as a cause or epiphenomenon of AMS and/or high-altitude pulmonary edema, remains to be elucidated.