Ultrastructural localization of collagen IV, fibronectin, and laminin in the trabecular meshwork of normal and glaucomatous eyes.

PURPOSE: To determine whether differences in the ultrastructural characteristics or composition of the basement membranes of the trabecular lamellae and Schlemm's canal exist in normal eyes and eyes with primary open-angle glaucoma (POAG). Basement membranes play key roles in the attachment of the overlying trabecular cells and Schlemm's canal cells. METHODS: Electron microscopy used in conjunction with immunogold labeling was used to examine the ultrastructure of the basement membranes in the trabecular meshwork and to determine the presence of collagen IV, laminin, and fibronectin in 6 normal eyes and 6 eyes with POAG. To determine which cells in the meshwork synthesized these molecules in situ hybridization was studied in an additional 8 normal eyes. RESULTS: No distinctive ultrastructural changes were found in the basement membranes of glaucomatous eyes, whether early or advanced disease, when compared with normal eyes. Label for all three proteins was present in the basement membranes of the trabecular lamellae, Schlemm's canal, and in scattered patches within the juxtacanalicular tissue. Laminin and fibronectin were most abundant in the periphery of the sheath material surrounding the elastic tendons in the juxtacanalicular tissue. In contrast to previously published light microscopic studies, no increase in fibronectin was found in glaucoma. Regions of the basement membrane of the canal underlying giant vacuoles were similar to regions without giant vacuoles in both appearance and labeling. In situ hybridization revealed that mRNA for all three proteins was present in most trabecular cells throughout the meshwork; no regional differences in cellular labeling within were observed. CONCLUSION: The ultrastructural characteristics and immunogold labeling of basement membranes were similar in normal and glaucomatous eyes; no additional structures were labeled in POAG eyes that were not also labeled in normal eyes. Label of the patches of amorphous fibrogranular material within the juxtacanalicular tissue suggests it is basement membrane in origin, while the sheath material which is known to accumulate in POAG was not heavily labeled and does not appear to be basement membrane in origin.

Disruption of the FG nucleoporin NUP98 causes selective changes in nuclear pore complex stoichiometry and function.

The NUP98 gene encodes precursor proteins that generate two nucleoplasmically oriented nucleoporins, NUP98 and NUP96. By using gene targeting, we have selectively disrupted the murine NUP98 protein, leaving intact the expression and localization of NUP96. We show that NUP98 is essential for mouse gastrulation, a developmental stage that is associated with rapid cell proliferation, but dispensable for basal cell growth. NUP98-/- cells had an intact nuclear envelope with a normal number of embedded nuclear pore complexes. Typically, NUP98-deficient cells contained on average approximately 5-fold more cytoplasmic annulate lamellae than control cells. We found that a set of cytoplasmically oriented nucleoporins, including NUP358, NUP214, NUP88, and p62, assembled inefficiently into nuclear pores of NUP98-/- cells. Instead, these nucleoporins were prominently associated with the annulate lamellae. By contrast, a group of nucleoplasmically oriented nucleoporins, including NUP153, NUP50, NUP96, and NUP93, had no affinity for annulate lamellae and assembled normally into nuclear pores. Mutant pores were significantly impaired in transport receptor-mediated docking of proteins with a nuclear localization signal or M9 import signal and showed weak nuclear import of such substrates. In contrast, the ability of mutant pores to import ribosomal protein L23a and spliceosome protein U1A appeared intact. These observations show that NUP98 disruption selectively impairs discrete protein import pathways and support the idea that transport of distinct import complexes through the nuclear pore complex is mediated by specific subsets of nucleoporins.

Antigen retrieval of basement membrane proteins from archival eye tissues.

Antigen retrieval (AR) methods can unmask tissue antigens that have been altered by fixation, processing, storage, or resin interactions. This is particularly important in the study of archival tissues, because primary fixatives and storage times may vary among specimens. We performed an electron microscopic study of basement membrane components of the aqueous humor drainage pathways from archival eye tissue. AR (heated citrate buffer, pH 6.0, LR White resin) increased the amount of label of collagen IV and fibronectin in tissue fixed in four different fixatives, including those containing glutaraldehyde. Labeling density was approximately doubled after AR for most fixatives, with the largest increase for tissues fixed in 4% paraformaldehyde/2% glutaraldehyde. Duration of storage time for archival tissues did not affect AR results. AR did not change the components of the extracellular matrix labeled; no "new" components were labeled after AR. We conclude that AR in citrate buffer can be used on selected extracellular matrix antigens to enhance label that would otherwise be lost due to fixation and storage.

Functional interdependence and colocalization of endothelial nitric oxide synthase and heat shock protein 90 in cerebral arteries.

Heat shock protein 90 (HSP90), an essential component of several signal transduction systems, participates in the activation of endothelial nitric oxide synthase (eNOS) in cells. The objective of the current study was to determine if HSP90 and eNOS were functionally interdependent and colocalized in the cerebral circulation. The authors used isometric force recording, cyclic 3'5'-guanosine monophosphate (cGMP) radioimmunoassay (RIA), and immunogold electron microscopy (EM) to study canine basilar artery. They found that geldanamycin (0.1 to 10 microg/mL), a selective HSP90 inhibitor, caused concentration-dependent contractions in arterial rings (n = 6 dogs). Contractions to geldanamycin were unaffected by a cyclooxygenase inhibitor, indomethacin (10 micromol/L; P < 0.05, n = 6). Functional evidence for interaction between HSP90 and nitric oxide (NO)-mediated signaling included observations that the contractile effect of geldanamycin was the following: (1) endothelium-dependent, (2) abolished by Ng-nitro-L-arginine methylester (L-NAME; 0.3 mmol/L), and (3) non-additive with the contractile effect of this NOS inhibitor (P < 0.01, n = 6 for each). Furthermore, RIA showed significant reduction in cGMP levels in arteries treated with geldanamycin (3 microg/mL; P < 0.02, n = 8), whereas immunogold EM demonstrated areas of colocalization of HSP90 and eNOS selectively in the cytoplasm of endothelial cells. The current findings suggest that in cerebral arteries, endothelial HSP90 plays an important role in modulation of basal NO-mediated signaling. This interaction may be particularly important in stress-induced up-regulation of HSP90 with subsequent alteration of vasomotor function.

Adenovirus-mediated gene transfer to human cerebral arteries.

Gene therapy is being investigated as a putative treatment option for cardiovascular diseases, including cerebral vasospasm. Because there is presently no information regarding gene transfer to human cerebral arteries, the principal objective of this study was to characterize adenovirus-mediated expression and function of recombinant endothelial nitric oxide synthase (eNOS) gene in human pial arteries. Pial arteries (outer diameter 500 to 1,000 microm) were isolated from 30 patients undergoing temporal lobectomy for intractable seizures and were studied using histologic staining, histochemistry, electron microscopy, and isometric force recording. Gene transfer experiments were performed ex vivo using adenoviral vectors encoding genes for bovine eNOS (AdCMVeNOS) and Escherichia coli beta-galactosidase (AdCMVLacZ). In transduced arteries, studied 24 hours after exposure to vectors, expression of recombinant beta-galactosidase and eNOS was detected by histochemistry, localizing mainly to the adventitia (n = 4). Immunoelectron microscopy localized recombinant eNOS in adventitial fibroblasts. During contractions to U46619, bradykinin-induced relaxations were significantly augmented in AdCMVeNOS-transduced rings compared with control and AdCMVLacZ-transduced rings (P < 0.01; n = 6). The NOS inhibitor L-nitroarginine methylester (L-NAME) caused significantly greater contraction in AdCMVeNOS-transduced rings (P < 0.001; n = 4) and inhibited bradykinin-induced relaxations in control and transduced rings (P < 0.001; n = 6). The current findings suggest that in AdCMVeNOS-transduced human pial arteries, expression of recombinant eNOS occurs mainly in adventitial fibroblasts where it augments relaxations to NO-dependent agonists such as bradykinin. Findings from the current study might be beneficial in future clinical applications of gene therapy for the treatment or prevention of cerebral vasospasm.