Masamichi Ishizaki

Peritubular Capillary Regression during the Progression of Experimental Obstructive Nephropathy

Injury to the renal microvasculature may be a major factor contributing to the progression of renal disease. Although severe disruption of peritubular capillaries (PTC) could lead to marked tubulointerstitial scarring, elucidation of that process remains incomplete. This study investigated the morphologic changes in PTC and their likely regulation by vascular endothelial growth factor (VEGF) during the progression of tubulointerstitial injuries. Unilateral ureteral obstruction was induced in Wistar rats by ligation of the left ureter, and the kidneys were then collected at selected times. PTC lumina and the expression of VEGF and its receptor Flk-1 were immunohistochemically detected. Morphologic changes in PTC endothelial cells were examined by using Ki67 staining, terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling, and electron-microscopic studies. In the first week of the disease period, immunohistochemical labeling of tubular VEGF intensified, with accompanying deformation and dilation of adjacent thrombomodulin (TM)-positive PTC lumina; an angiogenic response of endothelial cells was demonstrated with Ki67 and TM double-staining. During the subsequent 2 wk, tubular VEGF labeling decreased until it was virtually absent, an effect confirmed by Western blotting. Concomitantly, labeling of the VEGF receptor Flk-1 in PTC endothelial cells decreased and PTC lumina began to regress, demonstrating endothelial cell apoptosis (as detected in terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling and electron-microscopic studies). By the end of week 4, the numbers of TM-positive PTC lumina were significantly decreased in areas of marked tubulointerstitial scarring. These results suggest that PTC regression, involving an early, unsustained, angiogenic response followed by progressive endothelial cell apoptosis, could be a potential factor contributing to tubulointerstitial scarring in this unilateral ureteral obstruction model.

Wound healing involves induction of cyclooxygenase-2 expression in rat skin.

Cyclooxygenase (COX), an enzyme essential for prostaglandin biosynthesis, has two isoforms, COX-1 and -2. We investigated temporal and spatial changes in localization of these two COX proteins and mRNAs after excisional injury in rat skin. We also quantified the expression of these proteins and studied the effects of a specific COX-2 inhibitor on healing. Immunohistochemistry and in situ hybridization respectively indicated that the COX-2 protein and mRNA were expressed mainly within the basal layer of the epidermis, peripheral cells in the outer root sheath of hair follicles, and fibroblast-like cells and capillaries near epidermal wound edges. Much less intense expression was observed in normal skin than in injured skin. Western analysis demonstrated marked induction of COX-2 protein beginning within 12 hours and peaking 3 days after injury. In contrast, localization of COX-1 protein and mRNA, as well as the amount of protein expression, showed no significant change during wound healing. Administration of the COX-2 inhibitor delayed re-epithelialization in the early phase of wound healing and also inhibited angiogenesis. Thus, COX-2 induction may be important in cutaneous wound healing.

Vascular Endothelial Growth Factor165 Resolves Glomerular Inflammation and Accelerates Glomerular Capillary Repair in Rat Anti–Glomerular Basement Membrane Glomerulonephritis

Vascular endothelial growth factor (VEGF) is essential for maintenance of the glomerular capillary network. The present study investigated the effects of VEGF in rats with progressive crescentic glomerulonephritis (GN). Necrotizing and crescentic GN was induced in rats by injection of anti-rat glomerular basement membrane (GBM) antibody. The alterations of glomerular capillaries and glomerular VEGF expression were assessed. In addition, the effects of continuous VEGF165 administration (10 microg/100 g per d) on glomerular capillaries, glomerular inflammation, and the course of crescentic GN were examined. The appropriate timing of VEGF administration in progressive GN also was evaluated. In anti-GBM GN, necrotizing and crescentic glomerular lesions occurred by day 7, and newly formed necrotizing lesions reoccurred by week 3. Expression of VEGF was markedly reduced in necrotizing and crescentic lesions. Capillary repair was impaired after capillary destruction in necrotizing and crescentic glomeruli, which rapidly progressed to sclerotic glomeruli with chronic renal failure. In contrast, in the rats that received VEGF165 administration from day 7, the necrotizing and crescentic lesions recovered and renal function significantly improved in week 4. This was evident by proliferating endothelial cells and glomerular capillary repair. In addition, VEGF administration decreased intercellular adhesion molecule-1 and monocyte chemoattractant protein-1 expression in glomeruli (particularly on endothelial cells), reduced glomerular infiltrating CD8-postive and ED-1-positive cells, and inhibited the newly formed necrotizing lesions. VEGF administration was apparently effective against both the inflammatory and necrotizing glomerular lesions. These results suggest that VEGF administration resolves glomerular inflammation and accelerates glomerular recovery in the progressive necrotizing and crescentic GN. The therapeutic application of VEGF may be clinically useful for severe GN accompanied by extensive glomerular inflammation and endothelial injury.

Role of elastic fiber degradation in emphysema-like lesions of pulmonary lymphangiomyomatosis

To study the pulmonary structural remodeling in pulmonary lymphangiomyomatosis, electron microscopy and light and electron microscopic immunohistochemical observations for elastin and alpha 1-antitrypsin were performed on five open lung biopsy samples. Lung specimens showed emphysema-like changes in areas of abnormally accumulated smooth muscle cells. In the alveolar walls having accumulated smooth muscle cells, elastic fibers were decreased in number, disrupted, granular, and occasionally accumulated. Ultrastructurally, elastic fibers in areas of smooth muscle cell accumulation showed poorly outlined amorphous components and a few microfibrils, and occasionally showed electron-dense granular deposits in and around the amorphous components. Spiraling collagen fibrils were frequently found associated with these abnormal elastic fibers. Immunohistochemistry for elastin showed even staining of amorphous components of elastic fibers in the areas of smooth muscle cell accumulation. alpha 1-Antitrypsin was also detected evenly in amorphous components of elastic fibers in the areas of smooth muscle cell accumulation. It is proposed that the emphysema-like lesions of lymphangiomyomatosis are mediated by the degradation of elastic fibers, and these degraded elastic fibers are related to an imbalance of the elastase/alpha 1-antitrypsin system similar to the probable pathogenesis of emphysema.

Role of MMP-2 in alveolar epithelial cell repair after bleomycin administration in rabbits.

Matrix metalloproteinases (MMPs) have been implicated in the pathological processes of interstitial lung diseases. However, underlying mechanisms, particularly for activity levels and distribution of activated MMP-2 in the disease process, are yet to be elucidated. The present study investigated the immunolocalization of MMP-2, membrane type 1-matrix metalloproteinase (MT1-MMP), tissue inhibitor of metalloproteinase (TIMP)-2, p53, and Ki-67 in a rabbit model of bleomycin-induced pulmonary fibrosis. Gelatin zymography and in situ zymography were used to examine the activity and the localization of MMP-2. Furthermore, we performed Western blot and in situ hybridization for MT1-MMP, an activator for MMP-2. The total MMP-2 level estimated by gelatin zymography increased significantly at 3, 7, and 14 days after bleomycin administration, compared with controls. In the immunohistochemical study, immunoreaction for MMP-2 was strongest in alveolar epithelial cells among the cell populations. Swollen and/or elongated type II alveolar epithelial cells showed strong immunoreactions for MMP-2, MT1-MMP, and TIMP-2. After bleomycin administration, immunoreaction for p53 was observed in bronchiolar and alveolar epithelial cells. The proportion of p53-positive cells was high in epithelial cells from 1 to 14 days as MMP-2 levels were increased, suggesting that p53 may be responsible, at least in part, for the increase of MMP-2. The ratio of activated MMP-2 to total MMP-2 estimated by gelatin zymography increased significantly at 3, 7, 14, and 28 days after bleomycin treatment. In situ zymography revealed that type II alveolar epithelial cells degraded gelatin. An increased expression of MT1-MMP protein was observed by Western blot following administration of bleomycin. In situ hybridization demonstrated that type II alveolar epithelial cells gave intense signal for MT1-MMP mRNA. These results suggest that type II alveolar epithelial cells express MT1-MMP and activate MMP-2 on their cell surfaces, which may lead to the elongation and migration of alveolar epithelial cells in the repair process of bleomycin-induced pulmonary fibrosis.

Corneal endothelial cell damage by free radicals associated with ultrasound oscillation.

OBJECTIVEnTo determine whether ultrasound oscillations in the anterior chamber cause corneal endothelial injury by free radicals.nnnMETHODSnA phacoemulsification probe was introduced into the anterior chamber of rabbits eyes through a limbal incision, and ultrasound oscillation was performed without emulsifying the lens. Rabbits were assigned to 4 treatment groups: (1) no treatment (controls); (2) only irrigation with a salt solution; (3) ultrasound only; and (4) ultrasound oscillations with a salt solution of 0.001M ascorbic acid. The corneas were immunohistochemically examined for oxidative stress using 8-hydroxy-2-deoxyguanosine (8-OHdG), apoptosis by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling (TUNEL) staining, and ultrastructural changes by electron microscopy. The lipid peroxide levels in the aqueous humor were also measured.nnnRESULTSnIn the ultrasound-only group, 8-OHdG-positive cells and TUNEL-positive cells were detected at 24 hours; necrotic cells were detected at 12 to 24 hours. Also, lipid peroxide levels were significantly increased at later times in the ultrasound group. Such changes were not observed in other groups.nnnCONCLUSIONnFree radicals induced by ultrasound oscillation can cause corneal endothelial damages. Clinical Relevance Clinicians should be aware that free radicals associated with ultrasound oscillation can injure the corneal endothelial cells.

Expression of K12 keratin in alkali-burned rabbit corneas

The healing of alkali-injured corneas is characterized by the persistence of polymorphonuclear leukocytes (PMN) in tissues and recurrent corneal epithelial defects. It has been suggested that the proteolytic enzymes secreted by PMN may account in part for the recurrent epithelial defects in the alkali-burned corneas. Cytoplasmic keratins, which form intracellular intermediate filaments, participate in the formation of hemidesmosomes and play a key role in the focal adhesion of epithelial cells to the basement membranes. The K3/K12 keratin pair is a major constituent of differentiated and stratified corneal epithelium. We have recently cloned the cDNA encoding the rabbit K12 keratin. In the present study we examined the expression of K12 keratin during the healing of alkali-burned rabbit corneas by slot-blot and in situ hybridization. Our results indicate that in normal cornea K12 keratin is equally expressed in all cell layers of stratified corneal epithelium and suprabasal layers of limbal epithelium, but not in bulbar conjunctival and other epithelia, i.e., lens, iris, and retinal pigment epithelium. The basal cells of the detached regenerating epithelium of the injured cornea express a very low level of K12 keratin. These observations are consistent with the notion that defective expression of K3/K12 keratins may play a role in the abnormal attachment of the regenerating epithelium to the basement membrane.

Role of matrix metalloproteinases and their tissue inhibitor of metalloproteinases in myxomatous change of cardiac floppy valves

To clarify the underlying cause of myxomatous changes in cardiac floppy valves, the expression of the matrix metalloproteinases (MMP) and the tissue inhibitors of metalloproteinases (TIMP) was investigated in cardiac valves. Valves were obtained from nine patients with floppy valves, from 13 patients with other valvular disease types, and from four patients with normal valves. Immunohistochemical analyses for MMP‐2, MMP‐9, TIMP‐1, and TIMP‐2, and gelatin zymography for MMP‐2 and MMP‐9 were performed. Compared with the spongiosa of normal valves, the myxomatous area of floppy valves had stronger immunohistochemical reaction to MMP‐2 and MMP‐9, and weaker reaction to TIMP‐2. Activated MMP‐2 and MMP‐9 were detected in eight out of nine cases of floppy valves. Activated MMP‐2 was detected at low levels in two cases of normal valves showing mild expansion of the spongiosa without macroscopic floppiness. The ratio of active/total MMP‐2 and MMP‐9 increased in floppy valves compared with normal valves. These results suggest that the imbalance between MMP and TIMP and the increased activity of MMP‐2 and MMP‐9 may correlate with myxomatous changes observed in floppy valves. Valves with a slight myxomatous change and activated MMP‐2 may develop into floppy valves with increases in the activity of MMP.

Glomerular Capillary Regeneration and Endothelial Cell Apoptosis in Both Reversible and Progressive Models of Glomerulonephritis

In summary, angiogenetic capillary regeneration with endothelial proliferation occurred among mesangiolytic lesions in Thy-1 GN, and the damaged glomerulus recovered its normal structure with the reconstruction of the capillary network. In anti-GBM GN on WKY rats, the damaged glomerulus showed rare capillary regeneration and progressed to global sclerosis. In Thy-1 GN, endothelial cell apoptosis was found in the regenerated capillaries with endothelial cell hypercellularity. On the other hand, in anti-GBM GN on WKY rats, the number of endothelial cell apoptosis increased during the evolution of glomerular sclerosis. We have concluded that glomerular capillary regeneration plays an essential role in the recovery of damaged glomeruli. Moreover, apoptosis is indispensable in regulating the number of intrinsic endothelial cells. We also found that endothelial apoptosis is important in progression of glomerular sclerosis.

APPEARANCE OF IMMUNE CELLS AND EXPRESSION OF MHC II DQ MOLECULE BY FIBROBLASTS IN ALKALI-BURNED CORNEAS

Corneal alkali burns are characterized by persistent inflammatory response and recurrent epithelial erosions. We examine whether immune cell types, i.e., T-cells and B-cells, play a role in this devastating process. Rabbit alkali-burned corneas that healed for 1—49 days were subjected to immunostaining with monoclonal antibodies (mAb) LI 1/135 (anti-T-cells), and 2C4 (anti-MHC II DQ). Serum was collected weekly and subjected to Western blot immunostaining to detect antibodies against denatured corneal proteins. Our observations demonstrated that all injured corneas reepithelialized within 3 days but then developed recurrent erosions. Immunohistochemical studies revealed that PMN, monocytes, and B-cells labeled by 2C4 mAb and T-cells labeled by LI 1/135 mAb appeared in the periphery of the cornea at 1 day after alkali burn. Many of these myeloid and lymphoid cells invaded the central stroma after 2 weeks of injuries when the alkali-burned corneas were heavily vascularized. In addition, some fibroblastic cells also expressed the MHC II DQ molecules in the alkali-burned corneas that had healed for <2 weeks. Plasma cells appeared in granulation tissue of injured corneas that had healed for <3 weeks. Western blot analysis demonstrated a production of heterogeneous antibodies in a majority of the rabbits (11 of 14) to various denatured corneal proteins (between 80 kDa and 25 kDa) at 5 weeks of alkali burn. Inflammatory cell types, i.e., PMN, macrophages could be found underneath the detached epithelium. These observations are consistent with the notion that the myeloid and lymphoid cells may participate in and complicate the healing of corneal alkali burns.