Proteasome Inhibition Increases the Efficiency of Lentiviral Vector-Mediated Transduction of Trabecular Meshwork.

Purpose: To determine if proteasome inhibition using MG132 increased the efficiency of FIV vector-mediated transduction in human trabecular meshwork (TM)-1 cells and monkey organ-cultured anterior segments (MOCAS). Methods: TM-1 cells were pretreated for 1 hour with 0.5% dimethyl sulfoxide (DMSO; vehicle control) or 5 to 50 µM MG132 and transduced with FIV.GFP (green fluorescent protein)- or FIV.mCherry-expressing vector at a multiplicity of transduction (MOT) of 20. At 24 hours, cells were fixed and stained with antibodies for GFP, and positive cells were counted, manually or by fluorescence-activated cell sorting (FACS). Cells transduced with FIV.GFP particles alone were used as controls. The effect of 20 µM MG132 treatment on high- and low-dose (2 × 107 and 0.8 × 107 transducing units [TU], respectively) FIV.GFP transduction with or without MG132 was also evaluated in MOCAS using fluorescence microscopy. Vector genome equivalents in cells and tissues were quantified by quantitative (q)PCR on DNA. Results: In the MG132 treatment groups, there was a significant dose-dependent increase in the percentage of transduced cells at all concentrations tested. Vector genome equivalents were also increased in TM-1 cells treated with MG132. Increased FIV.GFP expression in the TM was also observed in MOCAS treated with 20 µM MG132 and the high dose of vector. Vector genome equivalents were also significantly increased in the MOCAS tissues. Increased transduction was not seen with the low dose of virus. Conclusions: Proteasome inhibition increased the transduction efficiency of FIV particles in TM-1 cells and MOCAS and may be a useful adjunct for delivery of therapeutic genes to the TM by lentiviral vectors.

Caveolin-1 modulates intraocular pressure: implications for caveolae mechanoprotection in glaucoma.

Polymorphisms in the CAV1/2 genes that encode signature proteins of caveolae are associated with glaucoma, the second leading cause of blindness worldwide, and with its major risk factor, intraocular pressure (IOP). We hypothesized that caveolin-1 (Cav-1) participates in IOP maintenance via modulation of aqueous humor drainage from the eye. We localize caveolae proteins to human and murine conventional drainage tissues and show that caveolae respond to mechanical stimulation. We show that Cav-1-deficient (Cav-1-/-) mice display ocular hypertension explained by reduced pressure-dependent drainage of aqueous humor. Cav-1 deficiency results in loss of caveolae in the Schlemm's canal (SC) and trabecular meshwork. However, their absence did not appear to impact development nor adult form of the conventional outflow tissues according to rigorous quantitative ultrastructural analyses, but did affect cell and tissue behavior. Thus, when IOP is experimentally elevated, cells of the Cav-1-/- outflow tissues are more susceptible to plasma membrane rupture indicating that caveolae play a role in mechanoprotection. Additionally, aqueous drainage from Cav-1-/- eyes was more sensitive to nitric oxide (NO) synthase inhibition than controls, suggesting that excess NO partially compensates for outflow pathway dysfunction. These results provide a functional link between a glaucoma risk gene and glaucoma-relevant pathophysiology.

Formation of fenestrae in murine liver sinusoids depends on plasmalemma vesicle-associated protein and is required for lipoprotein passage.

Liver sinusoidal endothelial cells (LSEC) are characterized by the presence of fenestrations that are not bridged by a diaphragm. The molecular mechanisms that control the formation of the fenestrations are largely unclear. Here we report that mice, which are deficient in plasmalemma vesicle-associated protein (PLVAP), develop a distinct phenotype that is caused by the lack of sinusoidal fenestrations. Fenestrations with a diaphragm were not observed in mouse LSEC at three weeks of age, but were present during embryonic life starting from embryonic day 12.5. PLVAP was expressed in LSEC of wild-type mice, but not in that of Plvap-deficient littermates. Plvap(-/-) LSEC showed a pronounced and highly significant reduction in the number of fenestrations, a finding, which was seen both by transmission and scanning electron microscopy. The lack of fenestrations was associated with an impaired passage of macromolecules such as FITC-dextran and quantum dot nanoparticles from the sinusoidal lumen into Disse's space. Plvap-deficient mice suffered from a pronounced hyperlipoproteinemia as evidenced by milky plasma and the presence of lipid granules that occluded kidney and liver capillaries. By NMR spectroscopy of plasma, the nature of hyperlipoproteinemia was identified as massive accumulation of chylomicron remnants. Plasma levels of low density lipoproteins (LDL) were also significantly increased as were those of cholesterol and triglycerides. In contrast, plasma levels of high density lipoproteins (HDL), albumin and total protein were reduced. At around three weeks of life, Plvap-deficient livers developed extensive multivesicular steatosis, steatohepatitis, and fibrosis. PLVAP is critically required for the formation of fenestrations in LSEC. Lack of fenestrations caused by PLVAP deficiency substantially impairs the passage of chylomicron remnants between liver sinusoids and hepatocytes, and finally leads to liver damage.

The role of plasmalemma vesicle-associated protein (PLVAP) in endothelial cells of Schlemm's canal and ocular capillaries.

Plasmalemma vesicle-associated protein (PLVAP, PV-1) is an endothelial protein that specifically localizes to diaphragms of fenestrae in fenestrated capillaries, and to stomatal diaphragms of caveolae. Here we investigated the localization of PLVAP in Schlemm's canal endothelium and ocular capillaries, and studied the structural effects of PLVAP deficiency. In mouse, pig and human eyes, immunoreactivity for PLVAP was present in fenestrated capillaries of choroid and ciliary processes, but not in the continuous capillaries of retina and ciliary muscle. In all three species staining for PLVAP was seen in the endothelia of the outflow vessels of aqueous humor e.g. Schlemm's canal (SC, mouse and human), aqueous plexus (AP, pig) and the scleral collector channels. Essentially comparable findings were observed when the expression of ß-galactosidase was investigated in mutant heterozygous and homozygous PLVAP-deficient mice with LacZ inserted into the Plvap locus. By transmission electron microscopy, the vast majority of caveolae in SC endothelial cells showed a stomatal diaphragm. In addition, solitary fenestrae or minipores with a diaphragm were occasionally observed in SC or AP of all three species. In contrast, mutant Plvap(-/-) mice showed a complete absence of stomatal diaphragms in SC caveolae while no SC minipores were observed. Moreover, diaphragms were absent in fenestrae of endothelial cells in the capillaries of the ciliary processes or the choriocapillaris, findings which were associated with a substantial decrease in the number of fenestrae. PLVAP is expressed in endothelial cells of Schlemm's canal and is essential for the formation of diaphragms in vascular endothelial cells of the eye.

Lack of endothelial diaphragms in fenestrae and caveolae of mutant Plvap-deficient mice.

Plasmalemmal vesicle-associated protein (PLVAP, PV-1) is specifically expressed in endothelial cells in which it localizes to diaphragms of fenestrae, caveolae, and transendothelial channels. To learn about its function, we generated mutant mice that lack PLVAP. In a C57BL/6N genetic background, homozygous Plvap-deficient embryos die before birth and suffer from subcutaneous edema, hemorrhages, and defects in the vascular wall of subcutaneous capillaries. In addition, hearts of Plvap(-/-) embryos show ventricular septal defects and thinner ventricular walls. In wild-type embryos, PLVAP and caveolae with a stomatal diaphragm are present in endothelial cells of subcutaneous capillaries and endocardium, while a diaphragm is missing in caveolae of Plvap(-/-) littermates. Plvap(-/-) mice in a mixed C57BL/6N/FVB-N genetic background are born and survive at the most for 4 weeks. Capillaries of exocrine and endocrine pancreas and of kidney peritubular interstitium were investigated in more detail as examples of fenestrated capillaries. In these vascular beds, Plvap(-/-) mice show a complete absence of diaphragms in fenestrae, caveolae, and transendothelial channels, findings which are associated with a substantial decrease in the number of endothelial fenestrae. The changes in the capillary phenotype correlate with a considerable retardation of postnatal growth and anemia. Plvap(-/-) mice provide an animal model to clarify the specific functional role of endothelial fenestrae and their contribution to passage of water and solutes in different organs.