| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Regular Articles |
From the Schepens Eye Research Institute and the Department of
Ophthalmology,*
Harvard Medical School, Boston,
Massachusetts; and the Burnham Institute,
La
Jolla, California
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
The molecular basis for the apoptogenic effects of diabetes in the retina is not yet identified. Proteins of the Bcl-2 family are well known inducers and integrators of survival and death signals7 and may play a role in the cellular effects of diabetes. The pro-survival family members can inhibit apoptosis induced by a variety of cytotoxic insults, and the pro-apoptotic members mostly work by heterodimerizing with pro-survival proteins and antagonizing their effects, although the Bax group can also kill directly by damaging organelles.7 Hence, the relative concentration of these proteins is an important determinant of their final impact on cell fate, and their regulation is transcriptional as well as posttranslational.7 Decreased expression of the pro-survival molecule Bcl-X L lowers cellular tolerance to stress-induced apoptosis,8 and down-regulation of Bcl-2 is a concomitant of the apoptosis of thyroid follicular cells in human Hashimoto’s thyroiditis.9 Conversely, almost complete disappearance of pro-apoptotic Bax is a concomitant of human breast cancer.10,11 Of immediate relevance to diabetes, increased expression of Bax mediates enhancement of apoptosis in the pre-implantation blastocyst in a mouse model of diabetic embryopathy.12
In a previous study, we had observed that in the adult human retina Bcl-2 is expressed predominantly if not solely in Müller glial cells, and that its levels are not modified by diabetes.13 We have now examined the effects of diabetes on another pro-survival member of the family, Bcl-XL, and two pro-apoptotic members, Bcl-XS and Bax. Having detected increased Bax levels in the diabetic retinas, we examined whether they can be induced by hyperglycemia and are associated with the death of retinal pericytes, a hallmark of diabetic retinopathy.2
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Human eyes were obtained from certified eye banks through the National Disease Research Interchange. Criteria for acceptance or rejection of specimens were described previously.13,14 We studied eyes from a total of 18 diabetic (12 males and 6 females, age 67 ± 6 years, duration of diabetes 9 ± 4 years) and 20 nondiabetic donors (16 males and 4 females, age 65 ± 6 years). Duration of diabetes of less than 15 years was chosen to address mostly if not solely background, nonproliferative retinopathy.15 The time elapsed from death to enucleation was 3.5 ± 2 hours for the diabetic donors and 3 ± 1 hour for the nondiabetic donors. Eyes were either shipped unfixed to the laboratory on wet ice, or one of the eyes was fixed in 10% buffered formalin by the eye banks. The time to fixation was 11 ± 4 hours and the time to processing of the unfixed eyes 31 ± 7 hours for both the diabetic and nondiabetic specimens. The unfixed retinas were isolated and separated from the pigmented epithelium, and processed for extraction of total protein or isolation of retinal vessels. The fixed retinas were processed for preparation of frozen retinal sections13 or trypsin digests.5
Isolation of the Retinal Vascular Network
To examine vascular cells in situ, trypsin digests were prepared from fixed human retinas as described.5 To extract vascular proteins, unfixed retinas were cut in four quadrants and placed in distilled water (2 hours at 4°C) to permit osmotic lysis of the neural and glial elements.16 The preparation was then transferred to a 60-mm petri dish containing 200 µg (580 U) DNase I Type II (Sigma Chemical Co., St. Louis, MO) in 5 ml distilled water. During the incubation, the DNase solution was gently and repeatedly pipetted on the tissue until the preparation looked transparent (approximately 10 minutes.). The preparation was transferred back to water and the inner limiting membrane was gently pulled away under the dissecting microscope using a tweezers and keeping the vascular network flat and untangled. If required, remaining neural and glial elements were eliminated by further gentle agitation in distilled water. After microscopic verification of its purity, the vascular network was homogenized in lysis buffer (see below). In some preparations, two of the quadrants were mounted on silane-coated slides and fixed in 10% buffered formalin for immunohistochemical studies. Residual neural contamination of the isolated vascular preparations was titrated by comparing in Western blots the level of neuron-specific enolase (NSE) in 5 µg of vascular lysate with that in 5, 0.5, 0.25, and 0.1 µg of lysate prepared from the contralateral whole retina. Glial contamination was similarly assessed by comparing the level of glial fibrillary acidic protein (GFAP) in Western blots of vascular and total retinal lysates, and by performing GFAP immunohistochemistry on the isolated fixed vessels.
Culture of Retinal Pericytes
Bovine retinal pericytes were isolated and cultured as previously described.17 Briefly, bovine retinas were gently dissected and extensively washed with Dulbecco’s modified Eagle’s medium (DMEM) to eliminate residual pigment epithelium. Retinas were homogenized with one stroke of a hand-held homogenizer, and the homogenate was washed thoroughly with DMEM over an 88-µm mesh to dissociate the neural retina. The material remaining on the mesh was digested with 0.1% collagenase type 2 (Worthington Biochemical, Lakewood, NJ) for 1 hour at 37°C. The digested material was pipetted onto 88 µm mesh, and the small vessel fragments and single cells passing through the mesh were pelleted by centrifugation, washed 2x in DMEM, resuspended in complete medium, and plated in 100-mm culture dishes. Cultured retinal pericytes showed the characteristic variable size, stellate shape with multiple processes, and lack of confluency and of multiple layers. Endothelial cell contamination was tested with anti-von Willebrand factor antibodies and glial contamination with antibodies against GFAP. Cultures at first and subsequent passages were routinely found to be free of contaminants. Pericytes were grown in DMEM containing 5 mmol/L glucose and supplemented with 10% fetal calf serum, antibiotics, and antimycotics. All experiments were performed in second or third passage cells. Treatment with high glucose (25 mmol/L D-glucose added) was begun 5, 3, or 1 week before harvest; mannitol (25 mmol/L) was used as control for the effects of hypertonicity.
Western Blot Analysis
Retinas, isolated retinal microvessels, or cultured pericytes were homogenized in lysis buffer (30 mmol/L Tris-HCl, 10 mmol/L EGTA, 5 mmol/L EDTA, 1% Triton X-100, 250 mmol/L sucrose) containing 1 mmol/L NaF, 1 mmol/L phenylmethylsulfonyl fluoride, 15 µg/ml aprotinin, 5 µg/ml leupeptin, 5 µg/ml pepstatin, and 1 mmol/L Na3VO4. The homogenate was sonicated three times for 2 seconds and centrifuged at 16,000 x g for 15 minutes at 4°C. The supernatant was collected and stored at -80°C. The protein concentration of each lysate was determined with the Bradford method18 using bovine serum albumin (BSA) as the standard. The lysates were diluted 1:3 in Laemmli Sample Buffer (Bio-Rad, Hercules, CA) supplemented with 2-mercaptoethanol to a final concentration of 5%, and boiled for 5 minutes. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis, immunoblotting, and visualization of immunoreactive bands were performed as described previously.13 The primary antibodies and their concentrations were as follows: rabbit polyclonal anti-Bcl-X (Santa Cruz Biotechnology, Santa Cruz, CA) 0.5 µg/ml, mouse monoclonal anti-human NSE (Dako, Carpinteria, CA) 1 µg/ml, and rabbit polyclonal anti-GFAP (Dako) 0.1 µg/ml. Rabbit anti-human Bax antiserum 171211 and anti-Bcl-X antiserum 169519 were raised to synthetic peptides conjugated to ovalbumin. The antisera were thus pre-absorbed in 0.1 mol/L Tris buffer, pH 7.8, containing 0.1 mg/ml ovalbumin and 1% milk for 1 hour at 37°C before being applied to the membranes at a 1:1000 dilution.
Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR)
To confirm that the single Bax species identified in the human
retina could be designated as Bax 
, we performed RT-PCR using
primers encompassing intron 5, which is unspliced in Bax
ß.20
The primers were designed based on the GenBank
sequences of human Bax and Bax ß mRNAs (accession numbers L22473
and L22474, respectively). The forward primer TTG GAC TTC CTC CGG GAG
CG corresponded to bp 421–440 of exon 5, and the reverse primer CAG
TCC AAG GCA GCT GGG GC to bp 580–561 of exon 6. Bax forms would
yield a 180-bp fragment, whereas ß forms would yield a 810-bp
fragment. RT-PCR was performed as described previously14
using 35 cycles of amplification. The specificity of the 180-bp PCR
product was verified by digestion with BamHI and Bst O1,
which yielded fragments of the expected size.
Immunohistochemistry
Formalin-fixed retinal sections and vascular preparations were rehydrated in phosphate buffered saline (PBS) for 20 minutes, blocked with 2% BSA in PBS for 10 minutes, and incubated overnight in a moist chamber with the primary antibody diluted in PBS containing 2% BSA and 0.5% Triton X-100. For Bax and Bcl-X immunohistochemistry, the antisera were pre-absorbed in 1 ml of 0.1 mol/L Tris buffer, pH 7.8, containing 0.1% ovalbumin, 2% BSA, 2% goat serum, and 0.1% human serum for 1 hour at 37°C; and the histological preparations were pre-blocked in 0.1 mol/L Tris containing 2% BSA, 2% goat serum, and 0.5% Triton X-100 for 1 hour at room temperature. The pre-absorbed antisera were used at a 1:1000 dilution; negative controls received the same dilution of nonimmune rabbit serum pre-absorbed as the primary antiserum. GFAP antibodies were used at 0.8 µg/ml; the negative controls received an equivalent concentration of nonimmune rabbit IgG. After extensive washes in PBS, the preparations were reacted with the appropriate secondary antibodies and processed for immunofluorescence or peroxidase immunohistochemistry.14
Transferase-Mediated dUTP Nick End Labeling (TUNEL) Assay
To test whether intense Bax immunoreactivity in cells of human retinal vessels was associated with vascular cell apoptosis, retinal trypsin digests from three diabetic and four age-matched nondiabetic donors were tested with the terminal deoxynucleotidyl TUNEL assay (In situ Cell Death Detection Kit; Boehringer Mannheim Biochemicals, Indianapolis, IN) as described previously.5 On completion of the TUNEL reaction, the preparations were reacted with the Bax antiserum as described above, and Bax immunoreactivity was detected with a Cy3-conjugated secondary antibody. The green fluorescence of TUNEL-positive nuclei with chromatin fragmentation and the red fluorescence of Bax were observed under a Zeiss fluorescence microscope.
Annexin V Binding
Annexin V binding was used to detect early stages of apoptosis in bovine retinal pericytes cultured in high glucose. Cells that had been exposed to high glucose for 1, 3, or 5 weeks and companion cells cultured in normal glucose medium were trypsinized and processed following the ApoAlert Annexin V protocol (Clontech Laboratories, Palo Alto, CA). At least 20,000 events were analyzed by flow cytometry using a single laser emitting excitation light at 488 nm.
Statistical Analysis
The data are summarized with the mean ± SD. Statistical analysis was performed with the unpaired t-test for results obtained in human specimens and analysis of variance for those obtained in cultured cells.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
The yield of total retinal protein was identical in the diabetic
and control group (9.2 ± 2 mg per retina). Bax immunoblots
(Figure 1)
showed a single band of 22 to
23 kd, confirmed to correspond to Bax , the membrane form of
Bax,20
by RT-PCR analysis. The Bax signal was often
more pronounced in diabetic than in control specimens. In the group of
18 diabetic donors, retinal Bax levels were 40 ± 13 densitometric
units/µg protein versus 32 ± 9 in control donors
(P = 0.04). The interassay coefficient of
variation for the six samples tested in more than two blots was 6
± 5%, indicating excellent reproducibility. Bax levels did not
correlate with age or diabetes duration, nor with the time elapsed from
death to eye enucleation or processing.
|
, with specific staining
detected in cells of the inner nuclear layer, ganglion cells (and,
often, their nuclei), and the endothelial and medial layers of blood
vessels. There was no evidence of glial staining. The distribution was
similar in diabetic and control retinas, and differences in staining
intensity between the two groups could not be confidently appreciated.
|
yielded a doublet of proteins with an apparent molecular mass of 29 to
31 kd, and a smaller protein of ~19 kd. The doublet corresponds to
the long isoform of Bcl-X as reported in other tissues19
and is thought to reflect initiation from a cryptic translation
initiation codon in addition to the usual start site.21
The 19-kd band corresponds to
Bcl-XS.19
Diabetes did not change
retinal Bcl-X expression appreciably. Retinal levels of
Bcl-XL were 72 ± 34 densitometric
units/µg protein in the diabetic group and 62 ± 26 in the
control group (P = 0.3), and the levels of
Bcl-XS were 24 ± 18 and 19 ± 15,
respectively (P = 0.3).
|
Bax and Bcl-X in Human Retinal Microvessels
To obtain data on Bax and Bcl-X expression specific to
microvessels, we isolated the vascular tree from unfixed retinas using
hypotonic lysis. Microscopically, the vascular networks appeared free
of contamination and in Western blots showed a greater than 20-fold
enrichment in proteins such as occludin and endothelial nitric oxide
synthase unique to endothelial cells (data not shown). Bax appeared as
a single band in immunoblots of vessel lysates (Figure 4)
, and the amount of Bax per microgram
of protein was approximately half the amount measured in the whole
retinal lysate prepared from the contralateral retina. Diabetic donors
showing in the whole retina Bax levels greater than in nondiabetic
controls tested in the same assay also showed Bax levels greater than
controls in the microvessels of the contralateral retina (Figure 4)
. Of
the two Bcl-X isoforms, only Bcl-XL could be
detected in up to 20 µg of vascular lysates.
|
, glial contamination rendered inaccurate the estimation of vascular
protein, ie, the reference for quantitative comparison of Bax levels
between diabetic and nondiabetic vessels. Bax Expression and Pericyte Apoptosis in Situ
The pattern of Bax expression in retinal vessels could, however,
be examined in situ. This approach offered the opportunity
to also determine any relationship that focal increases in Bax
expression may have with apoptotic events. Retinal trypsin digests from
3 diabetic and 4 nondiabetic donors were studied for Bax
immunoreactivity after performing the TUNEL reaction. A faint granular
staining was present throughout the capillary bed (Figure 5)
, consistent with low basal expression
of Bax in these vessels, and was not obviously different in diabetic
and control specimens. In accordance with previous observations,
TUNEL-positive vascular cells were more numerous in diabetic (5 ±
4 per one-sixth of retina) than in nondiabetic (1 ± 1)
retinal vessels. Several TUNEL-positive pericyte nuclei in the diabetic
vessels were surrounded by bright Bax staining (Figure 5)
. The intense
red (Bax) and green (TUNEL) fluorescence colocalized in the same area,
but not within the same structures, suggesting increased cytoplasmic
Bax expression in cells with evidence of apoptotic nuclear
fragmentation. Only a few cells showed bright Bax staining as an
isolated feature, whereas some TUNEL-positive cells did not show Bax
staining. Of note, in a nondiabetic trypsin digest the only
TUNEL-positive pericyte nucleus was surrounded by intense Bax
immunoreactivity
|
To begin investigating mechanisms for Bax overexpression in
diabetic retinal cells, bovine retinal pericytes were cultured in the
presence of high glucose for several weeks. One week exposure to high
glucose did not appreciably alter Bax levels (118 ± 37% of
control). After 3 weeks of high glucose treatment (Figure 6)
, Bax levels had almost doubled
(1186 ± 270 densitometric units/µg protein versus
658 ± 364 in control cultures), and were significantly greater
than control values in pericytes exposed to high glucose for 5 weeks
(1469 ± 388 densitometric units/µg protein, P =
0.03). Hypertonicity induced by 25 mmol/L mannitol had no effect. At
the same time points, the levels of Bcl-XL were
not changed by high glucose (94 ± 1, 102 ± 1, and 95
± 12% of control, respectively), nor were the levels of
Bcl-XS (87 ± 3, 100 ± 4, and 96
± 2% of control).
|
.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
These observations advance the notion that diabetes induces an apoptogenic environment in the retina. Our original studies had proposed apoptosis as the mode of death of retinal vascular cells in human and experimental diabetes on the basis of nuclear chromatin morphology coupled with TUNEL.5 We sought supportive evidence and mechanistic insight by examining if diabetes alters the balance of endogenous regulators of apoptosis. Bcl-2, found almost exclusively in the retinal glia, was present at similar levels in diabetic and nondiabetic retinas.13 We have now observed that expression of another pro-survival molecule, Bcl-XL, which in the postmortem human retina localized mostly to blood vessels, is also apparently unchanged by diabetes, as is the expression of its pro-apoptotic isoform, Bcl-XS. Bax immunostaining was limited to the inner retina, and was prominent in ganglion cells and vascular cells, the cell types that are known to undergo accelerated apoptosis in diabetes.5,6 We found increased overall level of Bax in whole retinal lysates from diabetic donors indicating that in these retinas some cell populations, or some cells within given populations, were overexpressing Bax. This could not be readily appreciated in retinal sections immunostained for Bax owing to the limited quantitative capabilities of immunohistochemistry.
To examine if retinal vascular cells were among Bax overexpressing cells, we prepared isolated vascular networks. We discovered however, even in preparations that appeared pure microscopically, a sizable amount of glial contamination, which could not be accurately titrated and precluded Bax quantitation in vascular extracts. When we turned to analysis of the vessels in situ, we observed numerous focal increases in Bax staining localized around pericyte nuclei and often associated with TUNEL-positive fragmentation of the same nuclei. Similar colocalization22 or at least temporal association23,24 of Bax overexpression and TUNEL have been reported in cells of the central nervous system and the retina after experimental induction of transient ischemia or optic nerve lesions, which are well known stimuli of apoptotic death. Of course, the frequency of Bax- and TUNEL-positive cells was much greater in such acute experimental settings than in the diabetic retinal vessels, where our snapshots of apoptosis could only capture the few cells dying at that particular point in time within the chronic degenerative process. Of note, we did not encounter endothelial cells staining intensely for Bax in diabetic retinal vessels. This may reflect a different temporal relationship of Bax overexpression to apoptosis in endothelial cells and pericytes, or indicate that retinal endothelial cell death in diabetes occurs through mechanisms that do not involve Bax. The distribution of Bcl-2 family members as well as the regulation of apoptosis are often cell-specific within tissues.19,25 These possibilities require further investigation using model systems.
The observation that high glucose increases Bax levels in cultured pericytes at the same time as it increases in these cells the rate of apoptosis establishes that a metabolic abnormality characteristic of diabetes is sufficient to up-regulate Bax expression and stimulate a death pathway in retinal cells. Such notion could not be confidently derived from the findings in diabetic retina and retinal vessels in situ, where Bax expression may have been influenced by functional or structural sequelae of the chronic diabetes state, in addition to the prevailing abnormal milieu. That hyperglycemia may at least contribute to altering Bax levels in the diabetic retina is also suggested by the fact that high glucose in vitro selectively up-regulated Bax without affecting the levels of the two Bcl-X isoforms, exactly as observed in the diabetic retina. A role for hyperglycemia in inducing apoptosis mediated by increased Bax levels has been demonstrated in a model of diabetic embryopathy.12 The combined in vivo and in vitro data of our and Moley’s12 studies highlight once again molecular similarities in two diabetic pathologies, vascular disease and embryopathy, that are vastly different phenotypically. We had reported in the past that overexpression of extracellular matrix molecules, a hallmark of the microangiopathy that develops chronically in diabetes,26,27 also occurs within the rapid time frame of embryonic development in diabetic rats.28 The response to high glucose of both Bax and extracellular matrix molecules appears to be much slower and of lesser magnitude in adult cells29 (and this work) than in the developing embryo,12,28 possibly reflecting the different rates of metabolism and/or transcriptional activity in the two settings.
Increased Bax levels may play a role in the apoptosis of retinal cells by tilting the cellular balance of apoptosis regulators in a direction that increases susceptibility to stressful stimuli, but may also be sufficient to kill cells directly.7,30 The cultured pericyte model will permit us to address if glucose-induced Bax overexpression is the event responsible for pericyte apoptosis, and if so, what mechanisms are involved. In vivo studies would then be justified to test whether, in the absence of Bax, diabetes can still accelerate the death of vascular and neural cells in the retina and cause retinopathy.
|
Footnotes |
|---|
Supported by U.S. Public Health Service grant EY09122, a research grant of the American Diabetes Association, and the George and Frances Levin Endowment.
F. P. and G. R. contributed equally to this work.
Accepted for publication November 28, 1999.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
rken B: Overexpression of the death-promoting gene bax- which is downregulated in breast cancer restores sensitivity to different apoptotic stimuli and reduces tumor growth in SCID mice. J Clin Invest 1996, 97:2651-2659[Medline]
This article has been cited by other articles:
 |
|
 |
|
  M. Wu, Y. Chen, K. Wilson, A. Chirindel, M. A. Ihnat, Y. Yu, M. E. Boulton, L. I. Szweda, J.-X. Ma, and T. J. Lyons Intraretinal Leakage and Oxidation of LDL in Diabetic Retinopathy Invest. Ophthalmol. Vis. Sci., June 1, 2008; 49(6): 2679 - 2685. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. Liu, R. Yang, B. Tinner, A. Choudhry, N. Schutze, and B. Chaqour Cysteine-Rich Protein 61 and Connective Tissue Growth Factor Induce Deadhesion and Anoikis of Retinal Pericytes Endocrinology, April 1, 2008; 149(4): 1666 - 1677. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T Oshitari, S Yamamoto, N Hata, and S Roy Mitochondria- and caspase-dependent cell death pathway involved in neuronal degeneration in diabetic retinopathy Br. J. Ophthalmol., April 1, 2008; 92(4): 552 - 556. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. G. Miller, D. G. Smith, M. Bhat, and R. H. Nagaraj Glyoxalase I Is Critical for Human Retinal Capillary Pericyte Survival under Hyperglycemic Conditions J. Biol. Chem., April 28, 2006; 281(17): 11864 - 11871. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P Georges, E E Cornish, J M Provis, and M C Madigan Muller cell expression of glutamate cycle related proteins and anti-apoptotic proteins in early human retinal development Br. J. Ophthalmol., February 1, 2006; 90(2): 223 - 228. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. H. NAGARAJ, T. OYA-ITO, M. BHAT, and B. LIU Dicarbonyl Stress and Apoptosis of Vascular Cells: Prevention by {alpha}B-Crystallin Ann. N.Y. Acad. Sci., June 1, 2005; 1043(1): 158 - 165. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. M. Cacicedo, S. Benjachareowong, E. Chou, N. B. Ruderman, and Y. Ido Palmitate-Induced Apoptosis in Cultured Bovine Retinal Pericytes: Roles of NAD(P)H Oxidase, Oxidant Stress, and Ceramide Diabetes, June 1, 2005; 54(6): 1838 - 1845. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. S. Chang, A. N. Dale, and K. H. Moley Maternal Diabetes Adversely Affects Preovulatory Oocyte Maturation, Development, and Granulosa Cell Apoptosis Endocrinology, May 1, 2005; 146(5): 2445 - 2453. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Li, S. Telemaque, R. E. Miller, and J. D. Marsh High Glucose Inhibits Apoptosis Induced by Serum Deprivation in Vascular Smooth Muscle Cells via Upregulation of Bcl-2 and Bcl-xl Diabetes, February 1, 2005; 54(2): 540 - 545. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. A. Kowluru and S. Odenbach Effect of Long-Term Administration of {alpha}-Lipoic Acid on Retinal Capillary Cell Death and the Development of Retinopathy in Diabetic Rats Diabetes, December 1, 2004; 53(12): 3233 - 3238. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Z. Dagher, Y. S. Park, V. Asnaghi, T. Hoehn, C. Gerhardinger, and M. Lorenzi Studies of Rat and Human Retinas Predict a Role for the Polyol Pathway in Human Diabetic Retinopathy Diabetes, September 1, 2004; 53(9): 2404 - 2411. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. M. A. El-Asrar, L. Dralands, L. Missotten, I. A. Al-Jadaan, and K. Geboes Expression of Apoptosis Markers in the Retinas of Human Subjects with Diabetes Invest. Ophthalmol. Vis. Sci., August 1, 2004; 45(8): 2760 - 2766. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. A. Kowluru and S. N. Abbas Diabetes-Induced Mitochondrial Dysfunction in the Retina Invest. Ophthalmol. Vis. Sci., December 1, 2003; 44(12): 5327 - 5334. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A.-F. Li, T. Sato, R. Haimovici, T. Okamoto, and S. Roy High Glucose Alters Connexin 43 Expression and Gap Junction Intercellular Communication Activity in Retinal Pericytes Invest. Ophthalmol. Vis. Sci., December 1, 2003; 44(12): 5376 - 5382. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Zhang, C. Gerhardinger, and M. Lorenzi Early Complement Activation and Decreased Levels of Glycosylphosphatidylinositol-Anchored Complement Inhibitors in Human and Experimental Diabetic Retinopathy Diabetes, December 1, 2002; 51(12): 3499 - 3504. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Zhang, D. M. Rosenbaum, A. R. Shaikh, Q. Li, P. S. Rosenbaum, D. J. Pelham, and S. Roth Ischemic Preconditioning Attenuates Apoptotic Cell Death in the Rat Retina Invest. Ophthalmol. Vis. Sci., September 1, 2002; 43(9): 3059 - 3066. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. Romeo, W.-H. Liu, V. Asnaghi, T. S. Kern, and M. Lorenzi Activation of Nuclear Factor-{kappa}B Induced by Diabetes and High Glucose Regulates a Proapoptotic Program in Retinal Pericytes Diabetes, July 1, 2002; 51(7): 2241 - 2248. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S.-i. Yamagishi, Y. Inagaki, T. Okamoto, S. Amano, K. Koga, M. Takeuchi, and Z. Makita Advanced Glycation End Product-induced Apoptosis and Overexpression of Vascular Endothelial Growth Factor and Monocyte Chemoattractant Protein-1 in Human-cultured Mesangial Cells J. Biol. Chem., May 31, 2002; 277(23): 20309 - 20315. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Morikawa, P. Baluk, T. Kaidoh, A. Haskell, R. K. Jain, and D. M. McDonald Abnormalities in Pericytes on Blood Vessels and Endothelial Sprouts in Tumors Am. J. Pathol., March 1, 2002; 160(3): 985 - 1000. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Nakamura, A. J. Barber, D. A. Antonetti, K. F. LaNoue, K. A. Robinson, M. G. Buse, and T. W. Gardner Excessive Hexosamines Block the Neuroprotective Effect of Insulin and Induce Apoptosis in Retinal Neurons J. Biol. Chem., November 16, 2001; 276(47): 43748 - 43755. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. L. Gendron, W. V. Good, L. C. Adams, and H. Paradis Suppressed Expression of Tubedown-1 in Retinal Neovascularization of Proliferative Diabetic Retinopathy Invest. Ophthalmol. Vis. Sci., November 1, 2001; 42(12): 3000 - 3007. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Nakagami, R. Morishita, K. Yamamoto, S.-i. Yoshimura, Y. Taniyama, M. Aoki, H. Matsubara, S. Kim, Y. Kaneda, and T. Ogihara Phosphorylation of p38 Mitogen-Activated Protein Kinase Downstream of Bax-Caspase-3 Pathway Leads to Cell Death Induced by High D-Glucose in Human Endothelial Cells Diabetes, June 1, 2001; 50(6): 1472 - 1481. [Abstract] [Full Text]
|
|
|
|
|
|
C. Gerhardinger, K. D. McClure, G. Romeo, F. Podestà, and M. Lorenzi IGF-I mRNA and Signaling in the Diabetic Retina Diabetes, January 1, 2001; 50(1): 175 - 183. [Abstract] [Full Text]
|
|
|
|
|
|
T. S. Kern, J. Tang, M. Mizutani, R. A. Kowluru, R. H. Nagaraj, G. Romeo, F. Podesta, and M. Lorenzi Response of Capillary Cell Death to Aminoguanidine Predicts the Development of Retinopathy: Comparison of Diabetes and Galactosemia Invest. Ophthalmol. Vis. Sci., November 1, 2000; 41(12): 3972 - 3978. [Abstract] [Full Text]
|
|
| ||||||||||||
