The diaphragms of fenestrated endothelia: gatekeepers of vascular permeability and blood composition

Abstract

Fenestral and stomatal diaphragms are endothelial subcellular structures of unknown function that form on organelles implicated in vascular permeability: fenestrae, transendothelial channels, and caveolae. PV1 protein is required for diaphragm formation in vitro. Here, we report that deletion of the PV1-encoding Plvap gene in mice results in the absence of diaphragms and decreased survival. Loss of diaphragms did not affect the fenestrae and transendothelial channels formation but disrupted the barrier function of fenestrated capillaries, causing a major leak of plasma proteins. This disruption results in early death of animals due to severe noninflammatory protein-losing enteropathy. Deletion of PV1 in endothelium, but not in the hematopoietic compartment, recapitulates the phenotype of global PV1 deletion, whereas endothelial reconstitution of PV1 rescues the phenotype. Taken together, these data provide genetic evidence for the critical role of the diaphragms in fenestrated capillaries in the maintenance of blood composition.

Fig. 1. Lack of PV1 results in absence of endothelial diaphragms and decreased survival

A. Endogenous Pvlap/PV1 alleles deletion in all cell types via CMV-cre expression. B. PV1 mRNA levels in the liver of WT, PV1^+/− and PV1^−/− mice (n>4, stdev, **p <0.01). C. PV1, Cav1 and VE Cadherin protein levels shown by Western blotting of lung tissue from PV1^−/− mice and controls. D. Mouse survival at 28 days expressed as a percentage of the expected numbers (calculated assuming a mendelian distribution of offspring genotypes) of the indicated genotypes on either C57Bl/6 (white bars) or C57Bl/6-Balb/c-129Sv/J mixed background (solid bars). (p < 0.01, CHI²). E. Kaplan Mayer analysis of the survival rate of the WT (n=485), PV1^+/− (n=899) and PV1^−/− (n=92) mice on mixed background that survived past 28 days. F–Q. Transmission (F–K,O–P) and scanning (L–N, Q) electron micrographs from tissues of WT (+/+)(F,J,M,P) and PV1^−/− (−/−)(G–I,K,L,N,O,Q) mice. Transmission EM micrographs are from lung (F–G), adrenal (H), pancreas (I), kidney peritubular (J–K) and glomerular (O–P) ECs. The absence of caveolae SDs (black arrows) and of fenestrae FDs (red arrows) in PV1^−/− mice is indicated. Black arrowheads indicate the presence of caveolae SDs (F) and of fenestrae FDs (J) in WT tissues. The asterisk in J indicates a TEC with SDs. Scanning electron micrographs of liver sinusoidal fenestrae sieve plates (M-N), or increasing magnification (left to right) of kidney peritubular (L) or kidney glomerular (Q) capillaries. The right panels in O and Q demonstrate diaphragm less fenestrae at high surface density in PV1^−/− mice.

Fig. 2. Deletion of PV1 in endothelial cells but not hematopoietic cells phenocopies the full PV1 knockout

A. Schematic of targeted deletion of Plvap/PV1 gene in both endothelial and hematopoietic compartments via Tie2-cre and VE Cadherin-cre, and in the hematopoietic compartment only using Vav1-cre. (EC: endothelial cells) and (HC: hematopoietic cells) B–C. Western blotting of total lung membranes from PV1^ECKO-Tie2 (B) and PV1^HCKO-Vav mice (C) and controls with anti-PV1 antibodies. D. Mouse survival at 28 days expressed as a percentage of the expected numbers (calculated assuming a mendelian distribution of offspring genotypes) of the indicated genotypes on either C57Bl/6 (white bars) or C57Bl/6- 129Sv/J mixed background (solid bars) (p < 0.01, CHI²). E. Kaplan-Mayer analysis of the survival rate of PV1^L/L (n=274), PV1^ECKO-VEC (n=36), PV1^ECKO-Tie2 (n=79) and PV1^HCKO-Vav (n=22) on C57Bl/6-129Sv/J mixed background that survived past 28 days. PV1^−/− (n=92) mice were also plotted as reference. F. Electron micrographs demonstrating the absence of diaphragms in PV1^ECKO-Tie2 (a,d–f,f′,i–k) and PV1^ECKO-VEC (b–c,g,h) mice (arrows) and their presence in the PV1^HCKO-Vav1 (l) mice (arrowheads). Images are from pancreas (a,i), intestine (b), kidney (c,e–g, l), lung (d), adrenals (h, j), and liver (k). (k, scanning electron micrograph).

Fig. 3. PV1^−/− mice gradually develop a severe hypoproteinemia and hypertriglyceridemia

A–D. Total plasma protein (TP), albumin (Alb) levels and albumin/globulin ratio (A/G) in non-fasted 1 and 4 week-old (A, B), 24h fasted 10 week-old (C) PV1^−/− mice and 4 week-old PV1^ECKO-Tie2 mice (D) with control littermates (n>5, *p<0.05, **p<0.01) E. Coommassie Blue staining of a 7% SDS PAGE of equal volumes (1μl) of blood plasma collected from 1 (left) and 4 (right) week-old PV1^−/−, PV1^ECKO-Tie2 and control mice, as indicated. F. Equal volumes of serum or ascites fluid from 4 week-old PV1^−/−, WT, PV1^+/−, PV1^ECKO-Tie2, PV1^L/+, PV1^L/L and PV1^ECKO-VEC mice were subjected to agarose gel protein electrophoresis. Note that the ascites sample came from the PV1^−/− and PV1^ECKO-Tie2 mice whose serum is loaded on lanes 1 and 6 from left, respectively. Last two lanes on the right are reference human serum samples. G–H. Immunoglobulin A (IgA) and M (IgM) plasma levels in 4 week-old PV1^−/− versus control mice. (n>4–6, **p<0.01) I. Lithium heparin plasma obtained from PV1^−/− or PV1^ECKO mice (left) has a lipid-rich, milky appearance as compared to plasma of WT, PV1^+/−, PV1^L/L or PV1^HCKO mice (right). J. Electron micrographs showing the presence of lipophylic lipid particles (arrow) in the plasma of kidney peritubular capillary (left) of a 28 day-old PV1^−/− mouse. At 8 weeks of age lipids (asterisk) in the plasma may coalesce and fully plug capillaries (middle). At 10–12 weeks (right) lipid cuffs (asterisks) surround capillaries in many organs. K–M. Plasma lipid profiles in 4 week-old PV1^−/− (K), 8 week-old PV1^ECKO-Tie2 (L) mice and 24 hours fasted 4 week-old PV1^−/− mice (n>8, for K and n>5 for L,M, *p<0.05, **p<0.01). N. Size exclusion by fast protein liquid chromatography (FPLC) of serum from PV1^−/− and control littermates. Left: UV absorption profiles of FPLC fractions from PV1^−/− (orange), PV1^+/− (blue) and WT (green) serum. Elution peaks of human VLDL, LDL and HDL controls are also shown (black). Average CHOL (middle) and TG (right) concentration in serum FPLC fractions from PV1^−/− (orange) and control (black) mice. (FPLC: fast protein liquid chromatography; LDL: low density lipoprotein; VLDL: very low density lipoprotein; HDL: high density lipoprotein) O–P. Western blotting with an ApoB antibody recognizing both ApoB48 and ApoB100 apolipoproteins of (O) serum FPLC fractions and (P) total plasma samples from PV1^−/− and control mice. All error bars: stdev.

Fig. 4. Vascular leak of proteins in PV1^−/− organs provided with fenestrated endothelium

A. Comparison of wet/dry weight ratio of different organs from PV1^−/− and WT mice at 4 weeks of age. B. H&E stained sections of formalin fixed paraffin embedded small intestine (jejeunum) of 2 week- (2 left panels), 4 week- (middle 4 panels) and 8 week-old mice (right 2 panels). In the left and middle panels the bottom micrographs demonstrate the intestine from WT (wt) whereas the top micrographs show intestine from PV1^−/− (−/−) mice. Both micrographs on the right demonstrate the intestine from PV1^ECKO-Tie2 (ECKO) mice in two different cuts (top - section orthogonal to the intestine wall; bottom - section parallel to the intestinal wall through the intestinal villi). In both PV1^−/− and PV1^ECKO-Tie2 samples arrows point to edema. C–D. Retention of Evans Blue (EB) at 15min following administration of EB-serum albumin in organs of (C) 4 week-old PV1^−/− mice and (D) 10 week-old PV1^ECKO-Tie2 mice and controls (n=4, **p<0.01). E. Representative image of duodenum of PV1^−/− (up) and WT (down) mice at 15 min post EB administration. F. EB quantification in ascites fluid and intestine lumen of PV1^−/− mice at 5 minutes post EB administration (n=4, **p<0.01). G. Measurement of leakage of fluorophore-labeled tracers in the ascites fluid of PV1^−/− mice at 5 and 15min post EB administration (data expressed as arbitrary fluorescence units). (n>3, p<0.01 vs WT) H a–e. Representative electron micrographs documenting leakage of serum albumin-gold nanoparticles in the adrenals of PV1^−/− mice. Arrowheads indicate the position of 10nm (black), 15nm (blue) and 25nm (red) albumin-gold conjugates after 5min perfusion of the tracer mixture. a and b show lower magnification fields with details magnified in a′ and b′. b is a montage of two separate micrographs of adjacent fields. All error bars: stdev.

Fig. 5. Endothelial specific reconstitution of PV1 rescues the PV^−/− phenotype

A. Schematic of endothelial specific PV1 reconstitution in PV1^ECRC mice. B. Western blotting of equal amounts of lung membrane proteins with anti-PV1, anti-HA and anti-VE Cadherin antibodies. C. Survival of PV1^ECRC and control genotypes at weaning on a mixed background. The orange and blue arrows highlight the observed differences in of PV1^ECRC and PV1^−/− offspring numbers, respectively. D. Kaplan Mayer analysis of the survival of PV1^ECRC, PV1HA and PV1^−/− mice that survived past 4 week-old. E. Electron micrographs documenting the reconstitution of SDs and FDs in capillary endothelia of PV1^ECRC mice: kidney (a,a′–c), pancreas (d,e), lung (f,g), heart (h), intestine villus (i, i′), adrenal (j), thyroid (k,l), salivary glands (m), thyroid (n,o) and liver sinusoid (p) and liver centrolobular vein (q). FDs are indicated with arrowheads, SDs of TEC with white arrows whereas SCs of caveolae are indicated by asterisks. Insets show higher magnification of relevant details. F–G. Comparison in 6 month-old PV1^ECRC, PV1HA (HA) and WT mice of (F) body weights of the (n>5, stdev), (G) total plasma protein (TP), albumin (Alb) levels and albumin/globulin ratio (A/G) (n=4, **p<0.01). H. Plasma total cholesterol (CHOL), HDL cholesterol (HDLc) and triglycerides (TG) concentration of 6 week-old PV1^ECRC mice. (n=4, **p<0.01). All error bars: stdev.