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Unlike during development, blood vessels in the adult are generally thought not to require VEGF for normal function. However, VEGF is a survival factor for many tumor vessels, and there are clues that some normal blood vessels may also depend on VEGF. In this study, we sought to identify which, if any, vascular beds in adult mice depend on VEGF for survival. Mice were treated with a small-molecule VEGF receptor (VEGFR) tyrosine kinase inhibitor or soluble VEGFRs for 1-3 wk. Blood vessels were assessed using immunohistochemistry or scanning or transmission electron microscopy. In a study of 17 normal organs after VEGF inhibition, we found significant capillary regression in pancreatic islets, thyroid, adrenal cortex, pituitary, choroid plexus, small-intestinal villi, and epididymal adipose tissue. The amount of regression was dose dependent and varied from organ to organ, with a maximum of 68% in thyroid, but was less in normal organs than in tumors in RIP-Tag2-transgenic mice or in Lewis lung carcinoma. VEGF-dependent capillaries were fenestrated, expressed high levels of both VEGFR-2 and VEGFR-3, and had normal pericyte coverage. Surviving capillaries in affected organs had fewer fenestrations and less VEGFR expression. All mice appeared healthy, but distinct physiological changes, including more efficient blood glucose handling, accompanied some regimens of VEGF inhibition. Strikingly, most capillaries in the thyroid grew back within 2 wk after cessation of treatment for 1 wk. Our findings of VEGF dependency of normal fenestrated capillaries and rapid regrowth after regression demonstrate the plasticity of the adult microvasculature. vascular endothelial growth factor inhibitors; endothelial fenestration; immunohistochemistry; electron microscopy

The vasculature of the embryo requires vascular endothelial growth factor (VEGF) during development, but most adult blood vessels lose VEGF dependence. However, some capillaries in the respiratory tract and selected other organs of adult mice regress after VEGF inhibition. The present study sought to identify the sequence of events and the fate of endothelial cells, pericytes, and vascular basement membrane during capillary regression in mouse tracheas after VEGF signaling was blocked with a VEGF-receptor tyrosine kinase inhibitor AG-013736 or soluble receptor construct (VEGF Trap or soluble adenoviral VEGFR-1). Within 1 day, patency was lost and fibrin accumulated in some tracheal capillaries. Apoptotic endothelial cells marked by activated caspase-3 were present in capillaries without blood flow. VEGF inhibition was accompanied by a 19% decrease in tracheal capillaries over 7 days and 30% over 21 days. During this period, desmin/ NG2-immunoreactive pericytes moved away from regressing capillaries onto surviving vessels. Empty sleeves of basement membrane, left behind by regressing endothelial cells, persisted for about 2 wk and served as a scaffold for vascular regrowth after treatment ended. The amount of regrowth was limited by the number of surviving basement membrane sleeves. These findings demonstrate that, after inhibition of VEGF signaling, some normal capillaries regress in a systematic sequence of events initiated by a cessation of blood flow and followed by apoptosis of endothelial cells, migration of pericytes away from regressing vessels, and formation of empty basement membrane sleeves that can facilitate capillary regrowth. apoptosis; basement membrane; endothelial cells; platelet-derived growth factor; pericytes; trachea; vascular endothelial growth factor

Angiopoietin-1 (Ang- 1) is essential for remodeling of the primitive vascular plexus and recruitment of mural cells during embryonic development. In the adult vasculature, Ang-1 can reduce plasma leakage in inflammation, but the mechanism of this action is not well understood. In the present study, we determined the magnitude and cellular mechanism of the antileak effect of Ang-1 in the airways of mice. Intravenous injection of bradykinin resulted in leakage of fluorescent microspheres (diameter 25-1,000 nm) from tracheal venules. The leakage peaked in 3-4 min and resolved by 10 min. High-resolution confocal microscopy revealed the presence of focal gaps at intercellular junctions of leaky venules. Genetically engineered Ang-1*, delivered systemically by adenoviral transduction of the liver, reduced leakage of 500-nm microspheres after bradykinin by 69%. The reduction in leakage coincided with a decrease in number and size of endothelial gaps. The proportion of venular surface occupied by endothelial gaps decreased 61%. Microsphere leakage correlated strongly with gap number and size ([r.sup.2] = 0.89). Together the results suggest that Ang-1 reduces leakage from inflamed venules by restricting the number and size of gaps that form at endothelial cell junctions through effects on intracellular signaling, cytoskeleton, and junction-related molecules. confocal microscopy; immunohistochemistry; respiratory tract; vascular permeability

Matrix metalloproteinase-2 and -9 expression increases in Mycoplasma-infected airways but is not required for microvascular remodeling. Am J Physiol Lung Cell Mol Physiol 287: L307-L317, 2004. First published April 9, 2004; 10.1152/ajplung.00404.2003.--Murine Mycoplasma pulmonis infection induces chronic lung and airway inflammation accompanied by profound and persistent microvascular remodeling in tracheobronchial mucosa. Because matrix metalloproteinase (MMP)-2 and -9 are important for angiogenesis associated with placental and long bone development and skin cancer, we hypothesized that they contribute to microvascular remodeling in airways infected with M. pulmonis. To test this hypothesis, we compared microvascular changes in airways after M. pulmonis infection of wild-type FVB/N mice with those of MMP-[9.sup.-/-] and MMP-[2.sup.-/-]/MMP-[9.sup.-/-] double-null mice and mice treated with the broad-spectrum MMP inhibitor AG3340 (Prinomastat). Using zymography and immunohistochemistry, we find that MMP-2 and MMP-9 rise strikingly in lungs and airways of infected wild-type FVB/N and C57BL/6 mice, with no zymographic activity or immunoreactivity in MMP-[2.sup.-/-]/MMP-[9.sup.-/-] animals. However, microvascular remodeling as assessed by Lycopersicon esculentum lectin staining of whole-mounted tracheae is as severe in infected MMP-[9.sup.-/-], MMP-[2.sup.-/-]/MMP-[9.sup.-/-] and AG3340-treated mice as in wild-type mice. Furthermore, all groups of infected mice develop similar inflammatory infiltrates and exhibit similar overall disease severity as indicated by decrease in body weight and increase in lung weight. Uninfected wild-type tracheae show negligible MMP-2 immunoreactivity, with scant MMP-9 immunoreactivity in and around growing cartilage. By contrast, MMP-2 appears in epithelial cells of infected, wild-type tracheae, and MMP-9 localizes to a large population of infiltrating leukocytes. We conclude that despite major increases in expression, MMP-2 and MMP-9 are not essential for microvascular remodeling in M. pulmonis-induced chronic airway inflammation. angiogenesis; Mycoplasma pulmonis; matrix metalloproteinase; MMP-2; MMP-9; AG3340; Prinomastat

Favre, Cecile J., Michael Mancuso, Kevin Maas, John W. McLean, Peter Baluk, and Donald M. McDonald. Expression of genes involved in vascular development and angiogenesis in endothelial cells of adult lung. Am J Physiol Heart Circ Physiol 285: H1917-H1938, 2003. First published July 3, 2003; 10.1152/ajpheart.00983.2002.--Profiling gene expression in endothelial cells advances the understanding of normal vascular physiology and disease processes involving angiogenesis. However, endothelial cell purification has been challenging because of the difficulty of isolating cells and their low abundance. Here we examine gene expression in endothelial cells freshly isolated from lung capillaries after in vivo labeling with fluorescent cationic liposomes and purification by fluorescence-activated cell sorting (FACS). Of the 39,000 genes and expressed sequence tags evaluated on custom oligonucleotide arrays, 555 were enriched in endothelial cell fraction. These included familiar endothelial cell-associated genes such as VEGF, VEGF receptor (VEGFR)-1, VEGFR-2, angiopoietin-2, Tiel, Tie2, Edg1 receptor, VE-cadherin, claudin 5, connexin37, CD31, and CD34. Also enriched were genes in semaphorin/ neuropilin (Sema3c and Nrp1), ephrin/Eph (ephrin A1, B1, B2, and EphB4), delta/notch (Hey1, Jagged 2, Notch 1, Notch 4, Numb, and Siah1b), and Wingless (Frizzled-4 and Tle1) signaling pathways involved in vascular development and angiogenesis. Expression of representative genes in alveolar capillary endothelial cells was verified by immunohistochemistry. Such expression reflects features that endothelial cells of normal lung capillaries have in common with embryonic and growing blood vessels. About half of the enriched genes, including exostosin 2, lipocalin 7, phospholipid scramblase 2, pleckstrin 2, protocadherin 1, Ryk, scube 1, serpinh1, SNF-related kinase, and several tetraspanins, had little or no previous association with endothelial cells. This approach can readily be used to profile genes expressed in blood vessels in tumors, chronic inflammation, and other sites in which endothelial cells avidly take up cationic liposomes. blood vessels; cationic liposomes; immunohistochemistry; gene profiling; oligonucleotide arrays

The expression of neurokinin-1 (NK1) receptors in the endothelial cells of angiogenic blood vessels which are hypersensitive to substance P was investigated. Pathogen-free male Fischer 344 rats were used as experimental models to assess whether there is an increase in the amount of substance P-containing nerves near the leaky blood vessels. Results showed an upregulation of NK1 receptors on endothelium as reflected in the increase in number of immunoreactive endosomes in angiogenic capillaries.

Researchers have investigated the fate of liposomes and liposome-DNA complexes injected intravenously. This was done by using CM-DiI, a red fluorescent lipophilic dye, to label cationic lipsome-DNA complexes which are known to promote effective gene transfer in a number of organs after intravenous injection into mice. It was found that CM-DiI-labeled DOTIM-cholesterol liposome-plasmid DNA complexes do not leak out of the vasculature in most organs. Instead, they adhere to and are internalized by endothelial cells in certain blood vessels, macrophages and intravascular leukocytes.

The effects of pulmonary inflammation on the number, morphology and size of endothelial gaps and plasma leakage were analyzed in the venules of rat tracheal mucosa after the intravenous injection of substance P. Substance P-mediated pulmonary inflammation and plasma leakage enhanced the density of vertical endothelial gaps and oblique slits. Furthermore, substance P-induced pulmonary leakage enhanced the formation of endothelial gaps.

The effects of inflammation on the binding pattern of different lectins to the vasculature of venules were analyzed in models of substance P-induced plasma leakage in the rat trachea light microscopy. Analysis of the selective binding of plant lectins to endothelial cell borders in inflamed venules of rat trachea indicated the presence of finger-like processes and endothelial gaps. The formation of endothelial gaps was caused by the deposition of dense reaction products in regions without cell retraction or endothelial gaps.

The effect of sensory denervation by capsaicin pretreatment at birth on immunologic response was investigated by measuring the number of Mycoplasma pulmonis organisms in the airway of rats. Capsaicin pretreatment increased the severity of the infection, as indicated by the increased thickness of the tracheal mucosa and the increase in substance P-induced plasma leakage. There was also a significant reduction in the number of SP-immunoreactive nerve fibers after capsaicin pretreatment and during infection. This was attributed to the increased destruction of sensory nerves during inflammation.

The relationship between substance P release and plasma leakage was investigated by examining the localization of neurokinin-1 receptors (NK1) in the rat tracheal mucosa after vagal stimulation. NK1 receptor-immunoreactive endosomes were found in the endothelial cells of venules and capillaries but not in the arterioles. Vagal stimulation increased the number of endosomes in the capillaries. The increase corresponded to the amount of plasma leakage. These effects were blocked by the NK1 receptor antagonist SR-140333.

The application of the scanning electron microscopy with backscattered and secondary electron imaging to study silver staining at endothelial cell borders of venules of the rat tracheal mucosa reveals that in normal venules the smooth contour of cell borders are followed by the silver lines. Endothelial cell borders are marked by silver nitrate. By staining an element of intercellular junctions, silver nitrate outlines endothelial cell gaps.

A study of the role of neurokinin type 1 (NK1) receptors in mediating tachykinin-induced adhesion of leukocytes to leaky venules in rats injected with substance P, NK1 receptor agonists or NK2 receptor agonists reveals that leukocyte adhesion and plasma leakage are mediated by NK1 receptors. Adhesion sites vary from leakage sites. Adherent neutrophils and eosinophils are related to each other in their numbers. Larger diameter venules exhibit greater extents of leukocyte adhesion than plasma leakage.

Beta2-adrenergic receptor agonist formoterol inhibits endothelial gap formation at plasma leakage sites to decrease the microvascular leakage. The receptor exhibits anti-edema effects and depends on the level of formoterol. The receptor decreases plasma extravasation by decreasing the number of gaps and not the gap size.

An enumeration of endothelial gaps in rat trachea venules and a linking of these values to the extent of plasma extravasation following an inflammatory stimulus confirms endothelial gaps at intercellular junctions as focal openings. These openings comprise less than 3% of the luminal surface and differ from the leukocyte attachment region. A reduction in the driving force for the connective shift of the tracer or a decline in the conductance of the gaps may be responsible for the decline in Monastral blue extravasation that occurs prior to closure of gaps.

Airway vasculature after mycoplasma infection: chronic leakiness and selective hypersensitivity to substance P. Am J Physiol Lung Cell Mol Physiol 280: L286-L297, 2001.-- Angiogenesis and microvascular remodeling are features of chronic airway inflammation caused by Mycoplasma pulmonis infection in rats. As airway blood vessels undergo remodeling, they become unusually sensitive to substance P-induced plasma leakage. Here we determined whether the remodeled vessels are leaky under baseline conditions, whether their heightened sensitivity is specific to substance P, and whether the leakage is reversible. Four weeks after infection, the amount of baseline leakage of Evans blue in the tracheal mucosa was two to five times the normal level. Gaps [is less than] 1 [micro]m in diameter were located between endothelial cells in some remodeled vessels. Substance P, but not platelet-activating factor or 5-hydroxytryptamine, produced an exaggerated leakage response. Inhalation of the [[Beta].sub.2]-adrenergic receptor agonist salmeterol reduced the leakage by [is less than] 60%. We conclude that the blood vessel remodeling after M. pulmonis infection is associated with microvascular leakiness due, in part, to the formation of endothelial gaps. This leakage is accompanied by an abnormal sensitivity to substance P but not to platelet-activating factor or 5-hydroxytryptamine and can be reduced by [[Beta].sub.2]-agonists. angiogenesis; [[Beta].sub.2]-adrenoceptor agonists; endothelial cells; Evans blue; inflammation; Mycoplasma pulmonis; salmeterol; vascular permeability; Wistar rats; Fischer 344 rats