Ultrastructural abnormalities of the trabecular meshwork extracellular matrix in Cyp1b1-deficient mice.

Cytochrome P450 1B1 (CYP1B1) is highly expressed in human and murine ocular tissues during development. Mutations in this gene are implicated in the development of primary congenital glaucoma (PCG) in humans. Mice deficient in Cyp1b1 (Cyp1b1(-/-) ) present developmental abnormalities similar to human primary congenital glaucoma. The present work describes the ultrastructural morphology of the iridocorneal angle of 21 eyes from 1-week-old to 8-month-old Cyp1b1(-/-) mice. Morphometric and semiquantitative analysis of the data revealed that 3-week-old Cyp1b1(-/-) mice present a significantly (P < .005) decreased amount of trabecular meshwork (TM) collagen and higher TM endothelial cell and collagen lesion scores (P < .005) than age-matched controls. Collagen loss and lesion scores were progressively increased in older animals, with 8-month-old animals presenting severe atrophy of the TM. Our findings advance the understanding of the effects of CYP1B1 mutations in TM development and primary congenital glaucoma, as well as suggest a link between TM morphologic alterations and increased intraocular pressure.

STAT1-mediated Bim expression promotes the apoptosis of retinal pericytes under high glucose conditions.

Hyperglycemia impacts different vascular cell functions and promotes the development and progression of various vasculopathies including diabetic retinopathy. Although the increased rate of apoptosis in pericytes (PCs) has been linked to increased oxidative stress and activation of protein kinase C-δ (PKC-δ) and SHP-1 (Src homology region 2 domain-containing phosphatase-1) tyrosine phosphatase during diabetes, the detailed mechanisms require further elucidation. Here we show that the rate of apoptosis and expression of proapoptotic protein Bim were increased in the retinal PCs of diabetic Akita/+ mice and mouse retinal PCs cultured under high glucose conditions. Increased Bim expression in retinal PCs under high glucose conditions required the sustained activation of signal transducer and activator of transcription 1 (STAT1) through production of inflammatory cytokines. PCs cultured under high glucose conditions also exhibited increased oxidative stress and diminished migration. Inhibition of oxidative stress, PKC-δ or Rho-associated protein kinase I/II was sufficient to protect PCs against apoptosis under high glucose conditions. Furthermore, PCs deficient in Bim expression were protected from high glucose-mediated increased oxidative stress and apoptosis. However, only inhibition of PKC-δ lowered Bim levels. N-acetylcysteine did not affect STAT1 levels, suggesting that oxidative stress is downstream of Bim. PCs cultured under high glucose conditions disrupted capillary morphogenesis of retinal endothelial cells (ECs) in coculture experiments. In addition, conditioned medium prepared from PCs under high glucose conditions attenuated EC migration. Taken together, our results indicate that Bim has a pivotal role in the dysfunction of retinal PCs under high glucose conditions by increasing oxidative stress and death of PCs.

Electrochemical evidence for the molten globule states of cytochrome c induced by N-alkyl sulfates at low concentrations.

The molten globule state (MG) of cytochrome c is the major intermediate of protein folding. The formation of MG state of cytochrome c is induced by n-alkyl sulfates such as sodium octyl sulfate (SOS), sodium dodecyl sulfate (SDS), and sodium tetradecyl sulfate (STS). The folding state of cytochrome c was monitored using circular dichroism (CD), isothermal titration calorimetry (ITC) and partial specific volumes. To explore a new approach for characterizing the MG conformation, cyclic voltametric studies of n-alkyl sulfates induced transition at acidic pH of cytochrome c (unfolded state, U) was carried out. Here, we have used a cystein-modified gold electrode, which is effective for direct rapid electron transfer to cytochrome c even in acid solutions, to directly observe electrochemistry in native (N) cytochrome c. Our results show that the extent of electron transfer is increased for U --> MG, and also the easiness of electron transferring occurred from MG --> N transition. Thus we demonstrate that the MG state of cytochrome c, induced by n-alkyl sulfates as salts with hydrophobic chains (hydrophobic salts), with different compactness reaches to near identical amount of electron transferring as N state.

Differential modulation of cadherin-mediated cell-cell adhesion by platelet endothelial cell adhesion molecule-1 isoforms through activation of extracellular regulated kinases.

The role of platelet endothelial cell adhesion molecule-1 (PECAM-1) in endothelial cell-cell interactions and its contribution to cadherin-mediated cell adhesion are poorly understood. Such studies have been difficult because all known endothelial cells express PECAM-1. We have used Madin-Darby canine kidney (MDCK) cells as a model system in which to evaluate the role of PECAM-1 isoforms that differ in their cytoplasmic domains in cell-cell interactions. MDCK cells lack endogenous PECAM-1 but form cell-cell junctions similar to those of endothelial cells, in which PECAM-1 is concentrated. MDCK cells were transfected with two isoforms of murine PECAM-1, Delta15 and Delta14&15, the predominant isoforms expressed in vivo. Expression of the Delta15 isoform resulted in apparent dedifferentiation of MDCK cells concomitant with the loss of adherens junctions, down-regulation of E-cadherin, alpha- and beta-catenin expression, and sustained activation of extracellular regulated kinases. The Delta15 isoform was not concentrated at cell-cell contacts. In contrast, the Delta14&15 isoform localized to sites of cell-cell contact and had no effect on MDCK cell morphology, cadherin/catenin expression, or extracellular regulated kinase activity. Thus, the presence of exon 14 in the cytoplasmic domain of PECAM-1 has dramatic effects on the ability of cells to maintain adherens junctions and an epithelial phenotype. Therefore, changes in the expression of exon 14 containing PECAM-1 isoforms, which we have observed during development, may have profound functional consequences.

Thrombospondin-1, a natural inhibitor of angiogenesis, is present in vitreous and aqueous humor and is modulated by hyperglycemia.

Negative regulators of angiogenesis play a major role in maintaining vascular homeostasis. Thrombospondin-1 (TSP1) is a natural inhibitor of angiogenesis. This report examines the presence of TSP1 in ocular samples and determines whether its production is altered in diabetes. Western blot analysis detected a 140 kDa antiangiogenic fragment of TSP1(gp140) in vitreous samples prepared from normal human and rat eyes. Intact TSP1 was detected in aqueous humor samples prepared from normal rat and bovine eyes. In contrast, TSP1 was virtually absent in vitreous and aqueous humor samples prepared from diabetic rat eyes. Furthermore, production of TSP1 by microvascular endothelial cells in culture was sensitive to high concentrations of glucose. Retinal blood vessels appeared nonuniform and dilated in diabetic animals when compared to control animals. These results demonstrate that TSP1 and its antiangiogenic fragment are present in aqueous humor and vitreous of normal rat eyes and are dramatically reduced in diabetes. Thus, TSP1 may play a role in ocular vascular homeostasis and its absence may contribute to vascular dysfunctions associated with diabetes.

Focal adhesion kinase, paxillin, and bcl-2: analysis of expression, phosphorylation, and association during morphogenesis.

Cell adhesive mechanisms which determine tissue architecture during morphogenesis are tightly regulated and have an impact on apoptosis, cell migration, proliferation, and differentiation. Bcl-2 is a death repressor that protects cells from apoptosis initiated by a variety of stimuli including loss of cell adhesion. Utilizing the kidney as a model of an organ that undergoes three-dimensional development we demonstrate that bcl-2 directly associates with paxillin. Focal adhesion kinase (FAK)(p125) and paxillin(p68) were highly expressed and tyrosine phosphorylated during development but declined to low levels following renal maturation (postnatal day 20) in normal mice. The decline in the expression of p125 FAK and p68 paxillin occurred together with an increase in specific cleavage products of lower molecular weights. Mice deficient in bcl-2 are born with renal hypoplasia and succumb to renal failure as a result of renal multicystic disease. In kidneys from postnatal day 20 bcl-2 -/- mice, tyrosine phosphorylation of p125 FAK and p68 paxillin was not down-regulated. However, the level of expression was similar to that of normal mice. These results demonstrate that the developmentally regulated expression and phosphorylation of FAK and paxillin, in the presence of bcl-2, is necessary for normal morphogenesis. The interaction of paxillin with bcl-2 during nephrogenesis may provide an alternative to integrin(s) signaling through paxillin/FAK thus bypassing the need for adhesion-mediated survival during three dimensional morphogenesis. Dev Dyn 1999;215:371-382.

Nuclear localization of beta-catenin and loss of apical brush border actin in cystic tubules of bcl-2 -/- mice.

Tight regulation of the rates of cell proliferation and apoptosis is critical for normal nephrogenesis. Nephrogenesis is profoundly affected by the loss of bcl-2 expression. Bcl-2-deficient (bcl-2 -/-) mice are born with renal hypoplasia and succumb to renal failure secondary to renal multicystic disease. Cell-cell and cell-matrix interactions impact tissue architecture by modulating cell proliferation, migration, differentiation, and apoptosis. E-cadherin mediates calcium-dependent homotypic cell-cell interactions that are stabilized by its association with catenins and the actin cytoskeleton. The contribution of altered cell-cell interactions to renal cystic disease has not been delineated. Cystic kidneys from bcl-2 -/- mice displayed nuclear localization of beta-catenin and loss of apical brush border actin staining. The protein levels of alpha-catenin, beta-catenin, actin, and E-cadherin were not altered in cystic kidneys compared with normal kidneys. Therefore, an altered distribution of beta-catenin and actin, in kidneys from bcl-2 -/- mice, may indicate improper cell-cell interactions interfering with renal maturation and contributing to renal cyst formation.

Tissue specific expression of alternatively spliced murine PECAM-1 isoforms.

PECAM-1 (CD31) is a cell adhesion molecule that is highly expressed at the sites of endothelial cell-cell contact and at lower levels on the surface of platelets and leukocytes. It is a member of the immunoglobulin gene superfamily and undergoes alternative splicing to generate several isoforms that differ only in their cytoplasmic domains. The tissue distribution of the expression of different PECAM-1 isoforms has not been previously defined. We have examined PECAM-1 expression in various mouse tissues and endothelial cells. PECAM-1 mRNA was highly expressed in lung, heart, and kidney, and to a lower extent in brain and liver. Most endothelial cells in culture expressed high levels of PECAM-1 mRNA; however, normal mouse brain endothelial cells rapidly lost PECAM-1 expression in culture. To examine the tissue distribution of PECAM-1 isoform expression, RT/PCR was performed on the RNA isolated from various mouse tissues and mouse endothelial cells. Cloning and sequencing of the cDNA products indicated that most tissues and endothelial cells expressed several PECAM-1 isoforms at different frequencies. The PECAM-1 isoform that lacks exons 14 and 15 was most frequently detected in all cases. A novel PECAM-1 isoform that lacks exons 12 and 14 was detected in brain. An antibody to the extracellular domain of PECAM-1 reacted with two major bands, at 130 kDa and 110-120 kDa, in lysates prepared from endothelial cells or kidneys at different stages of development. An antibody prepared against PECAM-1 exon 14, which reacts only with cytoplasmic domain of PECAM-1 isoforms that contain exon 14, failed to react with the major lower molecular weight form of PECAM-1 in these lysates. Therefore, PECAM-1 isoforms that lack exon 14 are expressed in endothelial cells and tissues in developmentally regulated fashion. These results illustrate that multiple PECAM-1 isoforms are expressed in various mouse tissues and endothelial cells. Understanding the distribution of PECAM-1 isoforms, and the identity of intracellular proteins with which they may interact, will help to elucidate the role of PECAM-1 in endothelial cell-cell interactions and morphogenesis.

The human plasminogen activator inhibitor type I gene promoter targets to kidney.

The plasminogen activator inhibitor type 1 (PAI-1) gene encodes the physiological inhibitor of tissue-type and urokinase-type plasminogen activators and is induced by cytokines such as transforming growth factor-beta (TGF-beta). Studies have identified DNA sequence elements within the first 1.3 kb of the 5'-upstream DNA that mediate cytokine responsiveness in transfected cells in vitro. However, the DNA sequences that mediate PAI-1 expression in vivo have not yet been delineated. To define these regulatory sequences, we generated transgenic mice that expressed a hybrid gene comprising sequences between -1,272 and +75 of the human PAI-1 gene ligated to a LacZ reporter gene. Transgene expression detected in two independent lines was observed only in kidney from embryonic day 13 to adult and was seen primarily in proximal tubule cells of the outer medulla. Transgene expression and activity were unchanged in response to TGF-beta and remained restricted to kidney. Thus we have identified a promoter region within the PAI-1 gene that targets transgene expression to kidney but, unlike the native promoter, is unresponsive to TGF-beta in the experimental protocol used.

Life, death and kidneys: regulation of renal programmed cell death.

Apoptosis plays an integral role during nephrogenesis and is tightly regulated by bcl-2. Transgenic mice manifesting a loss of bcl-2 expression demonstrate fulminant apoptosis of the metanephric blastema during kidney formation leading to renal hypoplasia at birth and multicystic renal disease later in life. In adult kidneys, the rate of apoptosis and level of bcl-2 expression are relatively low. Renal disease can alter the rate of apoptosis and/or elevate bcl-2 expression. The implications of such alterations are discussed.

Abnormal postpartum renal development and cystogenesis in the bcl-2 (-/-) mouse.

Mice deficient for B cell leukemia/lymphoma gene 2 [bcl-2(-/-) mice] manifest congenital renal hypoplasia and develop multicystic kidney disease and renal failure postnatally. To characterize postpartum renal development, to identify the cellular origin of the cysts, and to provide insight into the role that bcl-2 deficiency plays in the cystogenic process, we examined the morphology of kidneys from bcl-2 (-/-) mice and wild-type littermates [bcl-2 (+/+)] from birth (P0) to postpartum day 28 (P28), determined whether abnormalities of cellular proliferation and apoptosis accompany cyst development, and characterized expression of the bcl-2-related protein, bax. Between P0 and P7, kidneys from bcl-2 (-/-) and bcl-2 (+/+) mice undergo a comparable increase in weight and have similar histological appearances. However, during the next 2 wk of life, weight gain in kidneys from bcl-2 (-/-) mice is reduced compared with that in kidneys from bcl-2 (+/+) animals, and cysts develop in tubules with staining characteristics of proximal tubule, distal tubule/medullary thick ascending limb of Henle's loop, and collecting duct. Unaffected glomeruli and proximal tubules in kidneys of bcl-2 (-/-) mice undergo compensatory growth. Cystogenesis is accompanied by enhanced incorporation of 5-bromo-2'-deoxyuridine in cells within cortex and medulla and apoptosis of cells within cysts and in the renal interstitium. Bax protein is expressed in the distal tubule in kidneys of bcl-2 (+/+) and bcl-2 (-/-) mice and in some, but not all cysts. We conclude that abnormal regulation of DNA synthesis and apoptosis accompany cystogenesis in bcl-2 (-/-) mice during postpartum kidney development. Continued expression of bax could enhance apoptotic cell death.

Abnormal renal development in the Os/+ mouse is intrinsic to the kidney.

The oligosyndactylism (Os/+) mouse, is a genetic model for oligomeganephronic congenital renal hypoplasia. To define the abnormality in renal development and to determine whether the abnormality is kidney autonomous, we examined kidneys from newborn and 21- and 63-day-old Os/+ and wild-type (+/+) mice, obtained metanephric kidneys from embryonic day 12 (E12) Os/+ and +/+ embryos, and compared growth and development of the metanephroi in vitro. Kidneys from newborn Os/+ mice were smaller than those from newborn +/+ mice and contained fewer glomeruli per midsagittal section. Following birth, kidneys from Os/+ mice manifest compensatory growth of glomeruli and proximal tubules. Metanephroi from E12 Os/+ and +/+ embryos were comparable in size. However, during 4 days in culture, growth and development of metanephroi from Os/+ embryos were visibly reduced compared with metanephroi from +/+ embryos. Expression of B cell leukemia/lymphoma gene 2 (bcl-2), the absence of which is known to result in congenital renal hypoplasia, was detected in the Os/+ mouse kidneys. We conclude that the renal abnormality in Os/+ mice is intrinsic to the kidney and does not result from the absence of bcl-2 expression.

Ablation of bcl-2 gene expression decreases the numbers of oocytes and primordial follicles established in the post-natal female mouse gonad.

Oocyte loss, either directly through attrition (germ cell death) or indirectly through follicular atresia (somatic or granulosa cell death), is a fundamental event associated with defining the time of normal or premature reproductive senescence in females. Although apoptosis has been reported to function as the underlying mechanism responsible for death of both germ cells and somatic cells in the ovary, the final molecular steps which commit ovarian cells to death have not been fully elucidated. To examine if death repressor activity of the bcl-2 gene product is important for germ cell survival, we conducted studies using a Bcl-2 loss-of-function (bcl-2 -/-) transgenic mouse model. Histological analyses revealed that ovaries collected from bcl-2 -/- mice possessed numerous aberrantly formed primordial follicle-like structures containing a single layer of granulosa cells without an oocyte. Additionally, the total number of primordial follicles present which contained a healthy oocyte was markedly reduced in bcl-2 -/- mice as compared to heterozygote (bcl-2 -/+) or wild-type (bcl-2 +/+) mice, suggesting that expression of the bcl-2 death repressor gene is critical for endowment of a normal complement of germ cells and primordial follicles in the mammalian ovary.

Fulminant metanephric apoptosis and abnormal kidney development in bcl-2-deficient mice.

Apoptosis of the developing metanephric kidney plays an important role in renal organogenesis. The bcl-2 is an oncogene that inhibits apoptotic cell death in a variety of settings. The bcl-2 (-/-) mice complete embryonic development but, in contrast to bcl-2 (+/-) and bcl-2 (+/+) littermates, manifest growth retardation, hypopigmentation of hair, lymphoid apoptosis, abnormal kidney morphology, and renal failure postnatally. To provide insight into the mechanism for the latter abnormalities, we examined metanephric kidneys from bcl-2 (-/-), bcl-2 (+/-), and bcl-2 (+/+) mice, as well as embryonic day 12 (E12) mouse embryos, and compared growth and development of metanephroi in vitro. Kidneys from bcl-2 (+/-) mice developed normally. In contrast, development of kidneys from bcl-2 (-/-) mice was abnormal as reflected by a marked reduction of renal size in newborns compared with kidneys of bcl-2 (+/-) littermates. In addition, kidneys from bcl-2 (-/-) mice contained far fewer nephrons and had smaller nephrogenic zones. Although metanephroi obtained from E12 bcl-2 (+/-) and bcl-2 (-/-) mouse embryos were comparable in size, apoptosis of cells within metanephric blastemas of metanephroi from E12 bcl-2 (-/-) embryos was strikingly enhanced compared with that in blastemas of metanephroi from bcl-2 (+/-) embryos. During 3 days in culture, growth and development of metanephroi from bcl-2 (-/-) embryos were visibly reduced compared with those from bcl-2 (+/-) embryos.(ABSTRACT TRUNCATED AT 250 WORDS)

Bcl-2-deficient mice demonstrate fulminant lymphoid apoptosis, polycystic kidneys, and hypopigmented hair.

bcl-2-/-mice complete embryonic development, but display growth retardation and early mortality postnatally. Hematopoiesis including lymphocyte differentiation is initially normal, but thymus and spleen undergo massive apoptotic involution. Thymocytes require an apoptotic signal to manifest accelerated cell death. Renal failure results from severe polycystic kidney disease characterized by dilated proximal and distal tubular segments and hyperproliferation of epithelium and interstitium. bcl-2-/-mice turn gray with the second hair follicle cycle, implicating a defect in redox-regulated melanin synthesis. The abnormalities in these loss of function mice argue that Bcl-2 is a death repressor molecule functioning in an antioxidant pathway.

Analysis of herpes simplex virus-induced mRNA destabilizing activity using an in vitro mRNA decay system.

Most host mRNAs are degraded soon after infection of cells with herpes simplex virus type 1 (HSV-1). This early shutoff or early destabilization response is induced by a virion component, the virion host shutoff (vhs) protein. HSV-1 mutants, vhs1 and vhs-delta Sma, which produce defective or inactive vhs protein, fail to induce early shutoff. We have used an in vitro mRNA decay system to analyze the destabilization process. Polysomes from uninfected human erythroleukemia cells, used as a source of target mRNAs, were mixed with polysomes or with post-polysomal supernatant (S130) from HSV-1- or mock-infected murine erythroleukemia cells. Normally stable gamma-globin mRNA was destabilized by approximately 15-fold with S130 from wild-type virus-infected cells but was not destabilized with S130 from mock-infected cells or from cells infected with either of the two HSV mutants. The virus-induced destabilizing activity had no significant effect on the in vitro half-lives of two normally unstable mRNAs, histone and c-myc. No destabilizing activity was detected in polysomes from infected cells. We conclude that a virus-induced destabilizer activity can function in vitro, is located in the S130 of infected cells, and accelerates the decay rates of some, but not all, polysome-associated host mRNAs.

Reduction of corneal astigmatism at cataract surgery.

We studied the effect at three months of four transverse astigmatic keratotomy incisions (TAK) performed prior to phacoemulsification in 61 eyes on corneal astigmatism. The optical zone was varied in each case depending upon the magnitude of preoperative astigmatism. The eyes were compared to 105 control eyes in which no astigmatic incisions were performed to assess the estimated effect of the TAK incisions. Keratometry readings were taken preoperatively and three months postoperatively. Surgically induced astigmatism was measured using vector corrected astigmatism. Improvement in astigmatism was reported in all three optical zone groups, but the astigmatism was undercorrected in each. No complications affecting vision were reported, indicating that TAK may be a safe way to reduce postoperative astigmatism when combined with phacoemulsification.

Analysis of events associated with cell cycle arrest at G2 phase and cell death induced by cisplatin.

DNA is the accepted target for cisplatin, but recent evidence has shed doubt on DNA synthesis as the critical process. L1210/0 cells incubated for 2 hours with cisplatin progress to the G2 phase of the cell cycle and are arrested there for several days. They then either progress in the cell cycle or die. In cells that eventually die, total transcription, polyadenylated [poly(A)+] RNA synthesis, and protein synthesis were markedly inhibited only after 48 hours. Nicotinamide adenine dinucleotide (NAD) and adenosine triphosphate (ATP) levels decreased after 3 days. Cell membrane integrity was lost after 4 days. These results demonstrate that cells can be lethally damaged, yet continue to undergo apparently normal metabolic activities for several days. In a previous study, DNA double-strand breaks were detected after 1 day. We now show that by 2 days, breaks are visible as fragmentation in the nucleosome spacer regions of chromatin. This type of damage is consistent with cell death occurring by the process of apoptosis. Cell shrinkage and morphology were also consistent with this type of cell death. The slow cell death reported here appears to occur at the G2/M transition and may involve events that normally occur at this stage of the cell cycle. These results demonstrate the importance of DNA degradation as an early and possibly essential step in cell death.

Influence of cis-diamminedichloroplatinum(II) on DNA synthesis and cell cycle progression in excision repair proficient and deficient Chinese hamster ovary cells.

DNA has been implicated as the critical intracellular target for cis-diamminedichloroplatinum(II) (cis-DDP) action. Inhibition of DNA synthesis is a consequence of platination and has become accepted as the critical step in cis-DDP-induced toxicity. We have previously demonstrated that, following incubation with cis-DDP, murine leukemia L1210 cells progress through synthesis only to arrest in the G2 phase of the cell cycle. The G2 arrest was transient at low drug concentrations and was persistent at higher concentrations with a concomitant loss of viability. Chinese hamster ovary cell lines both proficient and deficient for DNA excision repair have been used to analyze the relationship between inhibition of DNA synthesis and toxicity and to determine whether DNA repair is necessary for cell cycle progression. Two repair-deficient cell lines were hypersensitive to cis-DDP and demonstrated a marked arrest in the G2 phase. The arrest was transient over only a small range of concentrations. At higher concentrations, the arrest was persistent and the cells subsequently died. Incorporation of [3H]thymidine into macromolecules demonstrated no inhibition of DNA synthesis while these cells progressed through the S phase. In contrast, at higher, but nontoxic, concentrations of cis-DDP, the repair-proficient cells exhibited inhibition of DNA synthesis while in S. At toxic concentrations, these cells also arrested in G2. Therefore, direct inhibition of DNA synthesis correlated only with the concentration of drug and not with the different sensitivities of the cell lines. Arrest of cells in G2 did correlate with toxicity. In every cell line, the appearance of G2-arrested cells preceded cell disintegration. It is proposed that the G2-arrested cells preceded cell disintegration. It is proposed that the G2 arrest results from the inability of the cells to transcribe genes required for passage into mitosis. Cells proficient in DNA repair can circumvent this arrest by repairing the damaged DNA and permitting transcription to proceed. These results support the hypothesis that inhibition of DNA synthesis is not the critical step in cis-DDP-induced cytotoxicity.