PV-1 is a component of the fenestral and stomatal diaphragms in fenestrated endothelia

PV-1 is a novel endothelial protein shown by immunocytochemical tests to be specifically associated with the stomatal diaphragms of caveolae in lung endothelium. Although the highest expression levels of both mRNA and protein are in the lung, PV-1 also has been found to be expressed in other organs. Using a specific antibody to the extracellular domain of PV-1, we have extended the survey on the presence of this protein at light and electron microscope level in several rat organs. Here we show that by immunofluorescence the antibody recognizes with high specificity the endothelium of the fenestrated peritubular capillaries of the kidney and those of the intestinal villi, pancreas, and adrenals. By immunolocalization at electron microscope level, the antibody recognizes specifically the diaphragms of the fenestrae and the stomatal diaphragms of caveolae and transendothelial channels in the endothelia of these vascular beds. No signal was detected in the continuous endothelium of the heart, skeletal muscle, intestinal muscularis, or brain capillaries or the nondiaphragmed fenestrated endothelium of kidney glomeruli. Taken together, our findings define the only antigen to be localized thus far in fenestral diaphragms. They also show that the stomatal diaphragms of caveolae and transendothelial channels and the fenestral diaphragms might be biochemically related, in addition to being morphologically similar structures.

Protection against hemorrhagic shock in mice genetically deficient in poly(ADP-ribose)polymerase

Hemorrhagic shock (HS) and resuscitation leads to widespread production of oxidant species. Activation of the enzyme poly(ADP-ribose) polymerase (PARP) has been shown to contribute to cell necrosis and organ failure in various disease conditions associated with oxidative stress. We tested the hypothesis whether PARP activation plays a role in the multiple organ dysfunction complicating HS and resuscitation in a murine model of HS and resuscitation by using mice genetically deficient in PARP (PARP−/−) and their wild-type littermates (PARP+/+). Animals were bled to a mean blood pressure of 45 mmHg (1 mmHg = 133 Pa) and resuscitated after 45 min with isotonic saline (2× volume of shed blood). There was a massive activation of PARP, detected by poly(ADP-ribose) immunohistochemistry, which localized to the areas of the most severe intestinal injury, i.e., the necrotic epithelial cells at the tip of the intestinal villi, and colocalized with tyrosine nitration, an index of peroxynitrite generation. Intestinal PARP activation resulted in gut hyperpermeability, which developed in PARP+/+ but not PARP−/− mice. PARP−/− mice were also protected from the rapid decrease in blood pressure after resuscitation and showed an increased survival time, as well as reduced lung neutrophil sequestration. The beneficial effects of PARP suppression were not related to a modulation of the NO pathway nor to a modulation of signaling through IL-6, which similarly increased in both PARP+/+ and PARP−/− mice exposed to HS. We propose that PARP activation and associated cell injury (necrosis) plays a crucial role in the intestinal injury, cardiovascular failure, and multiple organ damage associated with resuscitated HS.

DC-SIGNR, a DC-SIGN homologue expressed in endothelial cells, binds to human and simian immunodeficiency viruses and activates infection in trans

DC-SIGN, a C-type lectin expressed on the surface of dendritic cells (DCs), efficiently binds and transmits HIVs and simian immunodeficiency viruses to susceptible cells in trans. A DC-SIGN homologue, termed DC-SIGNR, has recently been described. Herein we show that DC-SIGNR, like DC-SIGN, can bind to multiple strains of HIV-1, HIV-2, and simian immunodeficiency virus and transmit these viruses to both T cell lines and human peripheral blood mononuclear cells. Binding of virus to DC-SIGNR was dependent on carbohydrate recognition. Immunostaining with a DC-SIGNR-specific antiserum showed that DC-SIGNR was expressed on sinusoidal endothelial cells in the liver and on endothelial cells in lymph node sinuses and placental villi. The presence of this efficient virus attachment factor on multiple endothelial cell types indicates that DC-SIGNR could play a role in the vertical transmission of primate lentiviruses, in the enabling of HIV to traverse the capillary endothelium in some organs, and in the presentation of virus to CD4-positive cells in multiple locations including lymph nodes.

Transgenic mice that overexpress the human trefoil peptide pS2 have an increased resistance to intestinal damage.

pS2 is a member of the trefoil peptide family, all of which are overexpressed at sites of gastrointestinal injury. We hypothesized that they are important in stimulating mucosal repair. To test this idea, we have produced a transgenic mice strain that expresses human pS2 (hpS2) specifically within the jejunum and examined the effect of this overexpression on proliferation and susceptibility to indomethacin-induced damage. A transgenic mouse was produced by microinjecting fertilized oocytes with a 1.7-kb construct consisting of rat intestinal fatty acid binding protein promoter (positions -1178 to +28) linked to full-length (490 bp) hpS2 cDNA. Screening for positive animals was by Southern blot analysis. Distribution of hpS2 expression was determined by using Northern and Western blot analyses and immunohistochemical staining. Proliferation of the intestinal mucosa was determined by assessing the crypt cell production rate. Differences in susceptibility to intestinal damage were analyzed in animals that had received indomethacin (85 mg/kg s.c.) 0-30 h previously. Expression of hpS2 was limited to the enterocytes of the villi within the jejunum. In the nondamaged intestine, villus height and crypt cell production rate were similar in transgenic and negative (control) litter mates. However, there was a marked difference in the amount of damage caused by indomethacin in control and transgenic animals in the jejunum (30% reduction in villus height in controls vs. 12% reduction in transgenic animals, P < 0.01) but the damage sustained in the non-hpS2-expressing ileal region was similar in control and transgenic animals. These studies support the hypothesis that trefoil peptides are important in stimulating gastrointestinal repair.

Mouse Col18a1 is expressed in a tissue-specific manner as three alternative variants and is localized in basement membrane zones.

We have isolated overlapping cDNAs encoding the N-terminal non-triple-helical region of mouse alpha 1(XVIII) collagen and shown that three different variants of alpha 1(XVIII) collagen exist. Each of the three variants shows characteristic tissue-specific expression patterns. Immunohistochemical studies show positive staining for alpha 1(XVIII) collagen along the basement membrane zones of vessels in the intestinal villi, the choroid plexus, skin, liver, and kidney. Thus, we conclude that alpha 1(XVIII) collagen may interact (directly or indirectly) with components in basement membrane zones or on the basal surface of endothelial/epithelial cells.

Loss of Apc heterozygosity and abnormal tissue building in nascent intestinal polyps in mice carrying a truncated Apc gene.

Mutations in the APC (adenomatous polyposis coli) gene appear to be responsible for not only familial adenomatous polyposis but also many sporadic cases of gastrointestinal cancers. Using homologous recombination in mouse embryonic stem cells, we constructed mice that contained a mutant gene encoding a product truncated at a 716 (Apc delta 716). Mendelian transmission of the gene caused most homozygous mice to die in utero before day 8 of gestation. The heterozygotes developed multiple polyps throughout the intestinal tract, mostly in the small intestine. The earliest polyps arose multifocally during the third week after birth, and new polyps continued to appear thereafter. Surprisingly, every nascent polyp consisted of a microadenoma covered with a layer of the normal villous epithelium. These microadenomas originated from single crypts by forming abnormal outpockets into the inner (lacteal) side of the neighboring villi. We carefully dissected such microadenomas from nascent polyps by peeling off the normal epithelium and determined their genotype by PCR: all microadenomas had already lost the wild-type Apc allele, whereas the mutant allele remained unchanged. These results indicate that loss of heterozygosity followed by formation of intravillous microadenomas is responsible for polyposis in Apc delta 716 intestinal mucosa. It is therefore unlikely that the truncated product interacts directly with the wild-type protein and causes the microadenomas by a dominant negative mechanism.