An Optimized Evans Blue Protocol to Assess Vascular Leak in the Mouse.

Vascular leak, or plasma extravasation, has a number of causes, and may be a serious consequence or symptom of an inflammatory response. This study may ultimately lead to new knowledge concerning the causes of or new ways to inhibit or treat plasma extravasation. It is important that researchers have the proper tools, including the best methods available, for studying plasma extravasation. In this article, we describe a protocol, using the Evans blue dye method, for assessing plasma extravasation in the organs of FVBN mice. This protocol is intentionally simple, to as great a degree as possible, but provides high quality data. Evans blue dye has been chosen primarily because it is easy for the average laboratory to use. We have used this protocol to provide evidence and support for the hypothesis that the enzyme neprilysin may protect the vasculature against plasma extravasation. However, this protocol may be experimentally used and easily adapted for use in other strains of mice or in other species, in many different organs or tissues, for studies which may involve other factors that are important in understanding, preventing, or treating plasma extravasation. This protocol has been extensively optimized and modified from existing protocols, and combines reliability, ease of use, economy, and general availability of materials and equipment, making this protocol superior for the average laboratory to use in quantifying plasma extravasation from organs.

Protection against vascular leak in neprilysin transgenic mice with complex overexpression pattern.

Neprilysin (NEP) is a cell surface metallopeptidase found in many tissues. Based mostly on pharmacological manipulations, NEP has been thought to protect blood vessels from plasma extravasation. We have suggested that NEP may protect against pulmonary vascular injury. However, these prior studies did not utilize mice which overexpress NEP. The aims of the present investigation were to develop and characterize doubly transgenic (DT) mice that overexpress NEP universally and conditionally, and to investigate the protective effect that overexpressed NEP may have against plasma extravasation in the vasculature. The duodenum, which is often used to assess vascular permeability, and in which the NEP protein was overexpressed in our DT mice two-fold, was selected as our experimental preparation. We found that substance P-induced plasma extravasation was decreased substantially (3.5-fold) in the duodenums of our doxycycline-treated DT mice, giving independent evidence of NEP's protective effects against plasma extravasation. Transgenic lung NEP protein was not stably expressed in the DT mice, so we were not able to test the effect of NEP overexpression in the lung. Although initially overexpressed nearly nine-fold at that site, pulmonary NEP protein overexpression eventually dissipated. Surprisingly, at a time when there was no lung transgenic NEP protein overexpression, lung NEP mRNA expression was still increased 23-fold, indicating that the expression defect probably is not transcriptional. These studies help to characterize our complex transgenic model of NEP overexpression and further demonstrate NEP's protective effects against plasma extravasation.

Loss-of-function thrombospondin-1 mutations in familial pulmonary hypertension.

Most patients with familial pulmonary arterial hypertension (FPAH) carry mutations in the bone morphogenic protein receptor 2 gene (BMPR2). Yet carriers have only a 20% risk of disease, suggesting that other factors influence penetrance. Thrombospondin-1 (TSP1) regulates activation of TGF-ß and inhibits endothelial and smooth muscle cell proliferation, pathways coincidentally altered in pulmonary arterial hypertension (PAH). To determine whether a subset of FPAH patients also have mutations in the TSP1 gene (THBS1) we resequenced the type I repeats of THBS1 encoding the TGF-ß regulation and cell growth inhibition domains in 60 FPAH probands, 70 nonfamilial PAH subjects, and in large control groups. We identified THBS1 mutations in three families: a novel missense mutation in two (Asp362Asn), and an intronic mutation in a third (IVS8+255 G/A). Neither mutation was detected in population controls. Mutant 362Asn TSP1 had less than half of the ability of wild-type TSP1 to activate TGF-ß. Mutant 362Asn TSP1 also lost the ability to inhibit growth of pulmonary arterial smooth muscle cells and was over threefold less effective at inhibiting endothelial cell growth. The IVS8+255 G/A mutation decreased and/or eliminated local binding of the transcription factors SP1 and MAZ but did not affect RNA splicing. These novel mutations implicate THBS1 as a modifier gene in FPAH. These THBS1 mutations have implications in the genetic evaluation of FPAH patients. However, since FPAH is rare, these data are most relevant as evidence for the importance of TSP1 in pulmonary vascular homeostasis. Further examination of THBS1 in the pathogenesis of PAH is warranted.

Decreased neprilysin and pulmonary vascular remodeling in chronic obstructive pulmonary disease.

RATIONALE: Studies with genetically engineered mice showed that decreased expression of the transmembrane peptidase neprilysin (NEP) increases susceptibility to hypoxic pulmonary vascular remodeling and hypertension; in hypoxic wild-type mice, expression is decreased early in distal pulmonary arteries, where prominent vascular remodeling occurs. Therefore, in humans with smoke- and hypoxia-induced vascular remodeling, as in chronic obstructive pulmonary disease (COPD), pulmonary activity/expression of NEP may likewise be decreased. OBJECTIVES: To test whether NEP activity and expression are reduced in COPD lungs and pulmonary arterial smooth muscle cells (SMCs) exposed to cigarette smoke extract or hypoxia and begin to investigate mechanisms involved. METHODS: Control and advanced COPD lung lysates (n = 13-14) were analyzed for NEP activity and protein and mRNA expression. As a control, dipeptidyl peptidase IV activity was analyzed. Lung sections were assessed for vascular remodeling and oxidant damage. Human pulmonary arterial SMCs were exposed to cigarette smoke extract, hypoxia, or H2O2, and incubated with antioxidants or lysosomal/proteasomal inhibitors. MEASUREMENTS AND MAIN RESULTS: COPD lungs demonstrated areas of vascular rarification, distal muscularization, and variable intimal and prominent medial/adventitial thickening. NEP activity was reduced by 76%; NEP protein expression was decreased in alveolar walls and distal vessels; mRNA expression was also decreased. In SMCs exposed to cigarette smoke extract, hypoxia, and H2O2, NEP activity and expression were also reduced. Reactive oxygen species inactivated NEP activity; NEP protein degradation appeared to be substantially induced. CONCLUSIONS: Mechanisms responsible for reduced NEP activity and protein expression include oxidative reactions and protein degradation. Maintaining or increasing lung NEP may protect against pulmonary vascular remodeling in response to chronic smoke and hypoxia.

Neprilysin null mice develop exaggerated pulmonary vascular remodeling in response to chronic hypoxia.

Neprilysin is a transmembrane metalloendopeptidase that degrades neuropeptides that are important for both growth and contraction. In addition to promoting carcinogenesis, decreased levels of neprilysin increases inflammation and neuroendocrine cell hyperplasia, which may predispose to vascular remodeling. Early pharmacological studies showed a decrease in chronic hypoxic pulmonary hypertension with neprilysin inhibition. We used a genetic approach to test the alternate hypothesis that neprilysin depletion increases chronic hypoxic pulmonary hypertension. Loss of neprilysin had no effect on baseline airway or alveolar wall architecture, vessel density, cardiac function, hematocrit, or other relevant peptidases. Only lung neuroendocrine cell hyperplasia and a subtle neuropeptide imbalance were found. After chronic hypoxia, neprilysin-null mice exhibited exaggerated pulmonary hypertension and striking increases in muscularization of distal vessels. Subtle thickening of proximal media/adventitia not typically seen in mice was also detected. In contrast, adaptive right ventricular hypertrophy was less than anticipated. Hypoxic wild-type pulmonary vessels displayed close temporal and spatial relationships between decreased neprilysin and increased cell growth. Smooth muscle cells from neprilysin-null pulmonary arteries had increased proliferation compared with controls, which was decreased by neprilysin replacement. These data suggest that neprilysin may be protective against chronic hypoxic pulmonary hypertension in the lung, at least in part by attenuating the growth of smooth muscle cells. Lung-targeted strategies to increase neprilysin levels could have therapeutic benefits in the treatment of this disorder.

Lung Krüppel-like factor (LKLF) is a transcriptional activator of the cytosolic phospholipase A2 alpha promoter.

Increased expression of cPLA2 (cytosolic phospholipase A2) has been shown to be the cause of tumorigenesis of NSCLC (non-small-cell lung cancer). Our laboratory has previously demonstrated that oncogenic forms of Ras increase transcription of cPLA2 in normal lung epithelial cells and NSCLC lines through activation of the ERK (extracellular-signal-regulated kinase) and JNK (c-Jun N-terminal kinase) MAPK (mitogen-activated protein kinase) family. We have also defined a minimal region of the cPLA2 promoter that is critical for this induction. To identify potential transcription factors that bind to this region and regulate expression, a yeast one-hybrid screen was performed with a rat lung cDNA library. Multiple members of the Krüppel family were identified, with LKLF (lung Krüppel-like factor) being isolated a number of times. Overexpression of LKLF in lung epithelial cells or Drosophila SL-2 cells increased cPLA2 promoter activity. Conversely, expression of a dominant negative form of LKLF inhibited induction of cPLA2 promoter activity by oncogenic Ras in normal lung epithelial cells and NSCLC. By electrophoretic mobility-shift assay analysis, it was found that LKLF bound to a GC-rich region of the cPLA2 promoter located between -37 and -30 upstream from the transcription start site. Expression of siRNA (small interfering RNA) directed against LKLF inhibited basal expression of cPLA2 in lung epithelial cells and blocked induction by H-Ras. In NSCLC, siRNA against LKLF co-operated with siRNA against Sp1 (stimulatory protein 1) to inhibit cPLA2 promoter activity. Finally, recombinant LKLF was a substrate for ERKs. These results indicate that LKLF is an important regulator of cPLA2 expression and participates in the induction of this protein, which is critical for increased eicosanoid production associated with lung tumorigenesis.