Search

Diabetic retinopathy, also defined as microvascular complication of diabetes mellitus, affects the entire neurovascular unit with specific aberrations in every compartment. Neurodegeneration, glial activation and vasoregression are observed consistently in models of diabetic retinopathy. However, the order and the severity of these aberrations varies in different models, which is also true in patients. In this study, we analysed rat models of diabetic retinopathy with similar phenotypes to identify key differences in the pathogenesis. For this, we focussed on intercellular junction-associated gene expression, which are important for the communication and homeostasis within the neurovascular unit. Streptozotocin-injected diabetic Wistar rats, methylglyoxal supplemented Wistar rats and polycystin-2 transgenic (PKD) rats were analysed for neuroretinal function, vasoregression and retinal expression of junction-associated proteins. In all three models, neuroretinal impairment and vasoregression were observed, but gene expression profiling of junction-associated proteins demonstrated nearly no overlap between the three models. However, the differently expressed genes were from the main classes of claudins, connexins and integrins in all models. Changes in Rcor1 expression in diabetic rats and Egr1 expression in PKD rats confirmed the differences in upstream transcription factor level between the models. In PKD rats, a possible role for miRNA regulation was observed, indicated by an upregulation of miR-26b-5p, miR-122-5p and miR-300-3p, which was not observed in the other models. In silico allocation of connexins revealed not only differences in regulated subtypes, but also in affected retinal cell types, as well as connexin specific upstream regulators Sox7 and miR-92a-3p. In this study, we demonstrate that, despite their similar phenotype, models for diabetic retinopathy exhibit significant differences in their pathogenic pathways and primarily affected cell types. These results underline the importance for more sensitive diagnostic tools to identify pathogenic clusters in patients as the next step towards a desperately needed personalized therapy.

Microglial activation is implicated in retinal vasoregression of the neurodegenerative ciliopathy-associated disease rat model (i.e., the polycystic kidney disease (PKD) model). microRNA can regulate microglial activation and vascular function, but the effect of microRNA-124 (miR-124) on retinal vasoregression remains unclear. Transgenic PKD and wild-type Sprague Dawley (SD) rats received miR-124 at 8 and 10 weeks of age intravitreally. Retinal glia activation was assessed by immunofluorescent staining and in situ hybridization. Vasoregression and neuroretinal function were evaluated by quantitative retinal morphometry and electroretinography (ERG), respectively. Microglial polarization was determined by immunocytochemistry and qRT-PCR. Microglial motility was examined via transwell migration assays, wound healing assays, and single-cell tracking. Our data showed that miR-124 inhibited glial activation and improved vasoregession, as evidenced by the reduced pericyte loss and decreased acellular capillary formation. In addition, miR-124 improved neuroretinal function. miR-124 shifted microglial polarization in the PKD retina from the pro-inflammatory M1 phenotype to the anti-inflammatory M2 phenotype by suppressing TNF-α, IL-1β, CCL2, CCL3, MHC-II, and IFN-γ and upregulating Arg1 and IL-10. miR-124 also decreased microglial motility in the migration assays. The transcriptional factor of C/EBP-α-PU.1 signaling, suppressed by miR-124 both in vivo (PKD retina) and in vitro (microglial cells), could serve as a key regulator in microglial activation and polarization. Our data illustrate that miR-124 regulates microglial activation and polarization. miR-124 inhibits pericyte loss and thereby alleviates vasoregression and ameliorates neurovascular function.

Rationale: Vasoregression secondary to glial activation develops in various retinal diseases, including retinal degeneration and diabetic retinopathy. Photoreceptor degeneration and subsequent retinal vasoregression, characterized by pericyte loss and acellular capillary formation in the absence diabetes, are also seen in transgenic rats expressing the polycystic kidney disease (PKD) gene. Activated Müller glia contributes to retinal vasodegeneration, at least in part via the expression of the soluble epoxide hydrolase (sEH). Given that an increase in sEH expression triggered vascular destabilization in diabetes, and that vasoregression is similar in diabetic mice and PKD rats, the aim of the present study was to determine whether sEH inhibition could prevent retinal vasoregression in the PKD rat. Methods: One-month old male homozygous transgenic PKD rats were randomly allocated to receive vehicle or a sEH inhibitor (sEH-I; Sar5399, 30 mg/kg) for four weeks. Wild-type Sprague-Dawley (SD) littermates received vehicle as controls. Retinal sEH expression and activity were measured by Western blotting and LC-MS, and vasoregression was quantified in retinal digestion preparations. Microglial activation and immune response cytokines were assessed by immunofluorescence and quantitative PCR, respectively. 19,20-dihydroxydocosapentaenoic acid (19,20-DHDP) mediated Notch signaling, microglial activation and migration were assessed in vivo and in vitro. Results: This study demonstrates that sEH expression and activity were increased in PKD retinae, which led to elevated production of 19,20-DHDP and the depression of Notch signaling. The latter changes elicited pericyte loss and the recruitment of CD11b+/CD74+ microglia to the perivascular region. Microglial activation increased the expression of immune-response cytokines, and reduced levels of Notch3 and delta-like ligand 4 (Dll4). Treatment with Sar5399 decreased 19,20-DHDP generation and increased Notch3 expression. Sar5399 also prevented vasoregression by reducing pericyte loss and suppressed microglial activation as well as the expression of immune-response cytokines. Mechanistically, the activation of Notch signaling by Dll4 maintained a quiescent microglial cell phenotype, i.e. reduced both the surface presentation of CD74 and microglial migration. In contrast, in retinal explants, 19,20-DHDP and Notch inhibition both promoted CD74 expression and reversed the Dll4-induced decrease in migration. Conclusions: Our data indicate that 19,20-DHDP-induced alterations in Notch-signaling result in microglia activation and pericyte loss and contribute to retinal vasoregression in polycystic kidney disease. Moreover, sEH inhibition can ameliorate vasoregression through reduced activity of inflammatory microglia. sEH inhibition is thus an attractive new therapeutic approach to prevent retinal vasoregression.

Progressive kidney diseases are often associated with scarring of the kidney’s filtration unit, a condition called focal segmental glomerulosclerosis (FSGS). This scarring is due to loss of podocytes, cells critical for glomerular filtration, and leads to proteinuria and kidney failure. Inherited forms of FSGS are caused by Rac1-activating mutations, and Rac1 induces TRPC5 ion channel activity and cytoskeletal remodeling in podocytes. Whether TRPC5 activity mediates FSGS onset and progression is unknown. We identified a small molecule, AC1903, that specifically blocks TRPC5 channel activity in glomeruli of proteinuric rats. Chronic administration of AC1903 suppressed severe proteinuria and prevented podocyte loss in a transgenic rat model of FSGS. AC1903 also provided therapeutic benefit in a rat model of hypertensive proteinuric kidney disease. These data indicate that TRPC5 activity drives disease and that TRPC5 inhibitors may be valuable for the treatment of progressive kidney diseases., National Institutes of Health (U.S.) (Grant DK095045), National Institutes of Health (U.S.) (Grant DK099465), National Institutes of Health (U.S.) (Grant DK103658), National Institutes of Health (U.S.) (Grant DK083511), National Institutes of Health (U.S.) (Grant DK093746)

Cardiac, neuronal and renal tubular epithelial cells are the most metabolically active cells in the body. Their fate depends largely on their mitochondria as the primary energy generating system which participates in the control of apoptosis, cell cycle and metabolism. Thus, mitochondrial dysfunction is a hallmark of many chronic diseases including diabetic nephropathy. A drop in mitochondrial bioenergetics efficiency is often associated with altered expression of respiratory chain complexes. Moreover, recent studies demonstrate that cellular proteins can shuttle to mitochondria and modify their function directly. Here we illustrate two mitochondria isolation protocols; one is recommended if the purity of the mitochondrial fraction is a priority such as if the mitochondrial localization of a protein has to be validated, the other if a high yield of intact functional mitochondria is required for functional studies and quantitative Western blotting. Next, we provide a detailed protocol for Western blotting of isolated mitochondria and renal cortex either to prove the purity of isolated fractions or to quantify complexes of the mitochondrial respiratory chain. We used this approach to identify classically cell membrane bound angiotensin II receptors in mitochondria and to study the effect of these receptors on mitochondrial function in early stages of diabetic nephropathy.

In the heart, secretory renin promotes hypertrophy, apoptosis, necrosis, fibrosis, and cardiac failure through angiotensin generation from angiotensinogen. Thus, inhibitors of the renin-angiotensin system are among the most potent drugs in the treatment of cardiac failure. Renin transcripts have been identified encoding a renin isoform with unknown targets and unknown functions that are localized to the cytosol and mitochondria. We hypothesize that this isoform, in contrast to secretory renin, exerts cardioprotective effects in an angiotensin-independent manner. Cells overexpressing cytosolic renin were generated by transfection or obtained from CX(exon2-9)renin transgenic rats. Overexpression of cytosolic renin reduced the rate of necrosis in H9c2 cardiomyoblasts and in primary cardiomyocytes after glucose depletion. These effects were not mediated by angiotensin generation since an inhibitor of renin activity did not influence the in vitro effects. siRNA-mediated knockdown of endogenous cytosolic renin increased the rate of necrosis and aggravated the pro-necrotic effects of glucose depletion. Isolated perfused hearts obtained from transgenic rats overexpressing cytosolic renin exhibited a 50% reduction of infarct size after ischemia-reperfusion injury. Cytosolic renin is essential for survival, both under basal conditions and during glucose starvation. The protective effects are angiotensin-independent and contrary to the known actions of secretory renin.A cytosolic isoform of renin with unknown functions is expressed in the heart. Cytosolic renin diminishes ischemia induced damage to the heart. The protective effects of cytosolic renin contradict the known function of secretory renin. The effects of cytosolic renin are not mediated via angiotensin generation. Renin-binding protein is a potential target for cytosolic renin.

Diabetic nephropathy correlates more closely to defective mitochondria and increased oxidative stress in the kidney than to hyperglycemia. A key driving factor of diabetic nephropathy is angiotensin II acting via the G-protein–coupled cell membrane type 1 receptor. The present study aimed to investigate the role of the angiotensin II type 2 receptor (AT2R) at the early stages of diabetic nephropathy. Using receptor binding studies and immunohistochemistry we found that the mitochondria in renal tubules contain high-affinity AT2Rs. Increased renal mitochondrial AT2R density by transgenic overexpression was associated with reduced superoxide production of isolated mitochondria from non-diabetic rats. Streptozotocin-induced diabetes (28 days) caused a drop in the ATP/oxygen ratio and an increase in the superoxide production of isolated renal mitochondria from wild-type diabetic rats. This correlated with changes in the renal expression profile and increased tubular epithelial cell proliferation. AT2R overexpression in tubular epithelial cells inhibited all diabetes-induced renal changes including a drop in mitochondrial bioenergetics efficiency, a rise in mitochondrial superoxide production, metabolic reprogramming, and increased proliferation. Thus, AT2Rs translocate to mitochondria and can contribute to reno-protective effects at early stages of diabetes. Hence, targeted AT2R overexpression in renal cells may open new avenues to develop novel types of drugs preventing diabetic nephropathy.

Connective tissue growth factor (CTGF, also named CCN2) plays an important role in the development of tubulointerstitial fibrosis, which most critically determines the progression to end-stage renal failure in autosomal-dominant polycystic kidney disease (ADPKD), the most common genetically caused renal disease. We determined CTGF expression in a well-characterized animal model of human ADPKD, the PKD/Mhm (cy/+) rat. Kidneys of 12 weeks old (cy/+) as well as (+/+) non-affected rats were analyzed for CTGF RNA and protein expression by RT-PCR, Northern and Western blot analyses, in situ hybridization, and IHC. Besides the established expression of CTGF in glomerular cells in kidneys of wild-type (+/+) animals, in (cy/+) rats, CTGF mRNA and protein were robustly expressed in interstitial, stellate-shaped cells, located in a scattered pattern underlying the cystic epithelium and in focal areas of advanced tubulointerstitial remodeling. Renal CTGF mRNA and protein expression levels were significantly higher in (cy/+) rats compared with their (+/+) littermates. Detection of CTGF expression in cells adjacent to cystic epithelium and in areas of marked fibrosis suggests a role in the local response to cyst development and indicates that CTGF may be a relevant factor contributing to tubulointerstitial fibrosis in polycystic kidney disease.

Vasoregression characterizes diabetic retinopathy in animal models and in humans. We have recently demonstrated that vasoregression is earlier initiated in a rat model of ciliopathy-induced retinal neurodegeneration (TGR rat). The aim was to assess the balance between vasoregressive effects of chronic hyperglycemia and photoreceptor degeneration on adult vascular remodelling. The retinas were analyzed at 4 and 9 months after streptozotocin-induced diabetes. Neurodegeneration was determined by quantitation of cell numbers and retinal layer thickness. Vasoregression was assessed by quantitative retinal morphometry in retinal digest preparations. Retinal VEGF levels were measured by ELISA. Glial activation, expression and location of HSP27 and phosphorylated HSP27 were evaluated by immunofluorescence staining. Unexpectedly, the numbers of acellular capillaries were reduced at both time points and led to fewer intraretinal microvascular abnormalities in late stage diabetic TGR. Concomitantly, inner nuclear layers (INLs) in diabetic TGR rats were protected from cell loss at both time points. Consequently, glial activation was reduced, but VEGF level was increased in diabetic TGR retinas. Expressions of HSP27 were upregulated in glia cells in the preserved INL of diabetic TGR. Chronic hyperglycemia preserves the microvasculature in the retinal model of neurodegeneration. Cell preservation in the retinal INL was associated with protective gene regulation. Together, these data indicate that diabetes can induce vasoprotection, in which retinal glia can play a particular role.

Background Autosomal dominant polycystic kidney disease (ADPKD) is one of the most common human inherited diseases. Modifier genes seem to modulate the disease progression and might therefore be promising drug targets. Although a number of modifier loci have been already identified, no modifier gene has been proven to be a real modifier yet. Methods Gene expression profiling of two substrains of the Han:SPRD rat, namely PKD/Mhm and PKD/US, both harboring the same mutation, was conducted in 36-day-old animals. Catechol-O-methyltransferase (Comt) was identified as a potential modifier gene. A 3-month treatment with tolcapone, a selective inhibitor of Comt, was carried out in PKD/Mhm and PKD/US (cy/+) animals. Results Comt is localized within a known modifier locus of PKD (MOP2). The enzyme encoding gene was found upregulated in the more severely affected PKD/Mhm substrain and was hence presumed to be a putative modifier gene of PKD. The treatment with tolcapone markedly attenuated the loss of renal function, inhibited renal enlargement, shifted the size distribution of renal cysts and retarded cell proliferation, apoptosis, inflammation and fibrosis development in affected (cy/+) male and female PKD/Mhm and PKD/US rats. Conclusions Comt has been confirmed to be the first reported modifier gene for PKD and tolcapone offers a promising drug for treating PKD.

Podocytes have a significant role in establishing selective permeability of the glomerular filtration barrier. Sustained renin–angiotensin–aldosterone system activation is crucial to the pathogenesis of podocyte injury, but the mechanisms by which angiotensin II modulates podocyte survival due to physiological or injurious stimuli remain unclear. Here, we used proteomic analysis to find new mediators of angiotensin II–induced podocyte injury. Antioxidant protein peroxiredoxin 2 expression was decreased in cultured podocytes stimulated with angiotensin II. Peroxiredoxin 2 was found to be expressed in podocytes in vivo, and its expression was decreased in the glomeruli of rats transgenic for angiotensin II type 1 receptors in a podocyte-specific manner, or in rats infused with angiotensin II. Downregulation of peroxiredoxin 2 in podocytes resulted in increased reactive oxygen species release, protein overoxidation, and inhibition of the Akt pathway. Both treatment with angiotensin II and downregulation of peroxiredoxin 2 expression led to apoptosis of podocytes. Thus, peroxiredoxin 2 is an important modulator of angiotensin II–induced podocyte injury.

Extracellular Yersinia pseudotuberculosis employs a type III secretion system (T3SS) for translocating virulence factors ( Yersinia outer proteins [Yops]) directly into the cytosol of eukaryotic cells. Recently, we used YopE as a carrier molecule for T3SS-dependent secretion and translocation of listeriolysin O (LLO) from Listeria monocytogenes . We demonstrated that translocation of chimeric YopE/LLO into the cytosol of macrophages by Yersinia results in the induction of a codominant antigen-specific CD4 and CD8 T-cell response in orally immunized mice. In this study, we addressed the requirements for processing and major histocompatibility complex (MHC) class II presentation of chimeric YopE proteins translocated into the cytosol of macrophages by the Yersinia T3SS. Our data demonstrate the ability of Yersinia to counteract exogenous MHC class II antigen presentation of secreted hybrid YopE by the action of wild-type YopE and YopH. In the absence of exogenous MHC class II antigen presentation, an alternative pathway was identified for YopE fusion proteins originating in the cytosol. This endogenous antigen-processing pathway was sensitive to inhibitors of phagolysosomal acidification and macroautophagy, but it did not require the function either of the proteasome or of transporters associated with antigen processing. Thus, by an autophagy-dependent mechanism, macrophages are able to compensate for the YopE/YopH-mediated inhibition of the endosomal MHC class II antigen presentation pathway for exogenous antigens. This is the first report demonstrating that autophagy might enable the host to mount an MHC class II-restricted CD4 T-cell response against translocated bacterial virulence factors. We provide critical new insights into the interaction between the mammalian immune system and a human pathogen.

The PKD/Mhm(cy/+) rat is a widely used animal model for the study of human autosomal dominant polycystic kidney disease, one of the most common genetic disorders, affecting one in 1000 individuals. We identified a new gene, Anks6, which is mutated (Anks6(p.R823W)) in PKD/Mhm(cy/+) rats. The evidence for a causal link between Anks6(p.R823W) and cystogenesis is still lacking, and the function of Anks6 is presently unknown. This study presents a novel transgenic rat model that overexpresses the mutated 2.8-kb Anks6(p.R823W) cDNA in the renal tubular epithelium. The transgenic Anks6(p.R823W) acts in a dominant-negative fashion and causes a predictable polycystic phenotype that largely mimics the general characteristics of the PKD/Mhm(cy/+) rats. Cyst development is accompanied by enhanced c-myc expression and continuous proliferation, apoptosis, and de-differentiation of the renal tubular epithelium as well as by a lack of translational up-regulation of p21 during aging. Using Northern blot analysis and in situ hybridization studies, we identified the first 10 days of age as the period during which transgene expression precedes and initiates cystic growth. Thus, we not only provide the first in vivo evidence for a causal link between the novel Anks6(p.R823W) gene mutation and polycystic kidney disease, but we also developed a new transgenic rat model that will serve as an important resource for further exploration of the still unknown function of Anks6.

Zusammenfassung Polyzystische Nierenerkrankungen (PKD) sind der häufigste genetische Grund für ein terminales Nierenversagen. Flüssigkeitsgefüllte Zysten bilden sich im Nierenparenchym und beeinträchtigen die Nierenfunktion mit zunehmender Anzahl und Größe, bis diese vollkommen zum Erliegen kommt. Seit mehreren Jahrzehnten werden Tiermodelle mit PKD für die Aufklärung der molekularen Mechanismen der Zystogenese verwendet. War man anfangs auf zufällige, durch Spontanmutationen aufgetretene Zystenmodelle angewiesen, eröffneten transgene und Knock-out-Technologien in den letzen 20 Jahren eine völlig neue Dimension, die molekularen Pathomechanismen der Zystogenese durch gezielte genetische Veränderungen im Erbgut aufzuklären. Nur mit der Hilfe von Tiermodellen konnte die Lokalisation von „Zystenproteinen“ in den Zilien und die Beteiligung zilienabhängiger Signalkaskaden in der Zystogenese gezeigt werden. Dieser Artikel gibt einen Überblick über die derzeit vorhandenen murinen Tiermodelle mit PKD.

OBJECTIVE— The mechanism underlying pericyte loss during incipient diabetic retinopathy remains controversial. Hyperglycemia induces angiopoietin-2 (Ang-2) transcription, which modulates capillary pericyte coverage. In this study, we assessed loss of pericyte subgroups and the contribution of Ang-2 to pericyte migration. RESEARCH DESIGN AND METHODS— Numbers of total pericytes and their subgroups were quantified in retinal digest preparations of spontaneous diabetic XLacZ mice. Pericytes were divided into subgroups according to their localization, their position relative to adjacent endothelial cells, and the expression of LacZ. The contribution of Ang-2 to pericyte migration was assessed in Ang-2 overexpressing (mOpsinhAng2) and deficient (Ang2LacZ) mice. RESULTS— Pericyte numbers were reduced by 16% (P < 0.01) in XLacZ mice after 6 months of diabetes. Reduction of pericytes was restricted to pericytes on straight capillaries (relative reduction 27%, P < 0.05) and was predominantly observed in LacZ-positive pericytes (−20%, P < 0.01). Hyperglycemia increased the numbers of migrating pericytes (69%; P < 0.05), of which the relative increase due to diabetes was exclusively in LacZ-negative pericytes, indicating reduced adherence to the capillaries (176%; P < 0.01). Overexpression of Ang-2 in nondiabetic retinas mimicked diabetic pericyte migration of wild-type animals (78%; P < 0.01). Ang-2 deficient mice completely lacked hyperglycemia-induced increase in pericyte migration compared with wild-type littermates. CONCLUSIONS— Diabetic pericyte loss is the result of pericyte migration, and this process is modulated by the Ang-Tie system.

SummaryAngiopoietin-2 (Ang2) is among the relevant growth factors induced by hypoxia and plays an important role in the initiation of retinal neovascularizations. Ang2 is also involved in incipient diabetic retinopathy, as it may cause pericyte loss. To investigate the impact of Ang2 on developmental and hypoxia-induced angiogenesis, we used a transgenic mouse line overexpressing human Ang2 in the mouse retina. Transgenic mice displayed a reduced coverage of capillaries with pericytes (-14 %; p

Background: The pathogenesis of Alzheimer’s disease (AD) is characterized by neuronal injury, activation of microglia and astrocytes, deposition of amyloid-beta and secondary vessel degeneration. In the polycystic kidney disease (PKD) rat model, we observed neuronal injury, microglial activation and vasoregression. We speculated that this neuroretinal degeneration shares important pathogenetic steps with AD. Therefore, we determined the activation of astrocytes and the accumulation of amyloid-beta in PKD retinae. Methods: Immunohistochemistry of PKD retinae for vimentin, carboxymethyllysin, beta-Amyloid 1-42, High-Mobility-Group-Protein B1 and amyloid protein precursor was performed. Results: Adjunct to astrocyte activation, accumulation of beta-Amyloid 1-42 and High-Mobility-Group-Protein B1 in astrocytes and around vessels of the superficial network was found in PKD retinae prior to the onset of vasoregression. Amyloid precursor protein was localized adjacent to the outer segment of photoreceptors in PKD and control rats. The parallel appearance of AD-related peptides indicates an alarmine based response to photoreceptor degeneration and secondary vasoregression. Conclusion: The model has broad overlap with AD and may be suitable to study beneficial pharmacological concepts. Copyright (c) 2012 S. Karger AG, Basel

The cloning of the PKD1 and PKD2 genes has led to promising new insight into the mechanisms that are responsible for cyst development in patients with autosomal dominant polycystic kidney disease. Although the dominant pattern of inheritance would argue for haploinsufficiency, a gain of function, or a dominant negative mechanism, there is good evidence that autosomal dominant polycystic kidney disease behaves like a recessive disease on a cellular level (two-hit mechanism of cystogenesis). For testing of whether other pathomechanisms in addition to the two-hit hypothesis can explain cyst formation, two transgenic rat lines that contain a truncated human polycystin-2 cDNA were generated. The protein product lacks almost the entire COOH-terminus and mimics mutations that frequently are found in patients. The transgene-encoded mRNA could be detected in multiple tissues of both transgenic lines, with the highest expression in the kidney. Both lines present with renal cysts that originate predominantly from the proximal tubule; in the tubular epithelial cells, the epitope-tagged mutant protein was detected in the brush border and in primary cilia. Further evidence of the involvement of primary cilia stems from the finding of retinal degeneration in the transgenic rats and from the fact that stably transfected LLC-PK(1) cells that inducibly produced the truncated polycystin-2 protein elaborated shorter cilia. Other experimental approaches, such as a knock-in strategy, will be necessary to validate these results, but this is the first preliminary evidence that cyst formation is due not only to somatic mutations.

Osteopontin is a multifunctional protein secreted by epithelial cells of various tissues. Its expression in the adult rat major salivary glands has not yet been studied. We examined osteopontin expression by immunohistochemistry using a well characterized monoclonal antibody. Submandibular glands of young adult male rats (70-100 days old) showed specific expression in secretion granules of granular duct cells but also in cells of the striated ducts and excretory duct. In the major sublingual as well as the parotid gland expression was found solely in the duct system. In addition, a few interstitial-like cells exhibiting very strong immunostaining for osteopontin could be found in either organ. Expression could neither be seen in acinar cells nor in cells of the intercalated ducts. Moreover, in submandibular glands of more aged rats (6- to 7-month old) which show well developed granular convoluted tubules, there was almost exclusive expression of osteopontin in granular duct cells as well as in some interstitial-like cells, but barely in the striated/excretory duct system. Western blot analysis of the submandibular gland showed a specific band migrating at approximately 74 kDa, detectable at both age stages. Osteopontin secreted fom granular duct cells may influence the composition of the saliva, e.g. thereby modulating pathways affecting sialolithiasis. Its expression in striated duct cells may also hint to roles such as cell-cell attachment or cell differentiation. The cell-specific expression detected in the rat major salivary glands differs in part from that reported in mice, human and monkey.

Angiotensin II (AngII) is a critical determinant of glomerular function involving both hemodynamic and pressure-independent effects that are insufficiently understood. A novel transgenic rat (TGR) model with overexpression of the human AngII type 1 receptor (hAT1) in podocytes was developed to study the consequences of an increased AT1 signaling on the structure and function of the glomerular filter. Use of the nephrin promoter to target the podocytes resulted in an expression of the hAT1 at a level roughly two times higher than the endogenous AT1 throughout life. All male TGR developed significant albuminuria starting at 8 to 15 wk of age; systolic BP was not elevated. More or less concurrently, structural changes at the glomerulus were encountered, starting with ubiquitous formation of pseudocysts at podocytes, followed by foot process effacement and local detachments. This damage progressed to nephron loss via the well known pathway typical for classic focal segmental glomerulosclerosis. The structural changes significantly correlated with age ( r 2 = 0.76) and urinary albumin excretion ( r 2 = 0.70). The data provide direct evidence that increased AT1 signaling in podocytes leads to protein leakage and structural podocyte damage progressing to focal segmental glomerulosclerosis.

Immunity to Toxoplasma gondii critically depends on TNFR type I-mediated immune reactions, but the precise role of the individual ligands of TNFR1, TNF and lymphotoxin-alpha (LTalpha), is still unknown. Upon oral infection with T. gondii, TNF(-/-), LTalpha(-/-), and TNF/LTalpha(-/-) mice failed to control intracerebral T. gondii and succumbed to an acute necrotizing Toxoplasma encephalitis, whereas wild-type (WT) mice survived. Intracerebral inducible NO synthase expression and-early after infection-splenic NO levels were reduced. Additionally, peritoneal macrophages produced reduced levels of NO upon infection with T. gondii and had significantly reduced toxoplasmastatic activity in TNF(-/-), LTalpha(-/-), and TNF/LTalpha(-/-) mice as compared with WT animals. Frequencies of parasite-specific IFN-gamma-producing T cells, intracerebral and splenic IFN-gamma production, and T. gondii-specific IgM and IgG titers in LTalpha(-/-) and TNF/LTalpha(-/-) mice were reduced only early after infection. In contrast, intracerebral IL-10 and IL-12p40 mRNA expression and splenic IL-2, IL-4, and IL-12 production were identical in all genotypes. In addition, TNF(-/-), LTalpha(-/-), and TNF/LTalpha(-/-), but not WT, mice succumbed to infection with the highly attenuated ts-4 strain of T. gondii or to a subsequent challenge infection with virulent RH toxoplasms, although they had identical frequencies of IFN-gamma-producing T cells as compared with WT mice. Generation and infection of bone marrow reconstitution chimeras demonstrated an exclusive role of hematogeneously produced TNF and LTalpha for survival of toxoplasmosis. These findings demonstrate the crucial role of both LTalpha and TNF for control of intracerebral toxoplasms.

Background: The role of AT1 receptors in myocardial ischemia/reperfusion injury is unclear. We, therefore, investigated the effects of the AT1 receptor antagonist irbesartan (Irb) in isolated hearts of selective myocardial AT1 overexpressing transgenic [transgenic(αMHC-hAT1)594–17] and Sprague–Dawley rats (SD) subjected to ischemia/reperfusion injury. Methods and results: Hearts of 4-week-old male SD or transgenic rats were isolated and perfused with Krebs–Henseleit buffer with or without 10 μM Irb in Langendorff mode. After 15 min of stabilization, pressure–volume curves were obtained and the hearts subjected to 20 min ischemia followed by 30 min reperfusion. A second set of pressure–volume curves was obtained thereafter. Left ventricular developed pressure (LVDP), end-diastolic pressure (LVEDP), total coronary flow (CF) and oxygen consumption (MVO2) were recorded continuously. Myocardial efficiency was derived from the slope of relations of MVO2 to pressure/volume area. After 20 min ischemia, LVEDP was significantly higher in transgenic than in SD (35.7±1.8 vs. 29.2±1.0 mmHg, P

The ankyrin repeat and sterile α motif (SAM) domain–containing six gene ( Anks6 ) is a candidate for polycystic kidney disease (PKD). Originally identified in the PKD/Mhm(cy/+) rat model of PKD, the disease is caused by a mutation (R823W) in the SAM domain of the encoded protein. Recent studies support the etiological role of the ANKS6 SAM domain in human cystic diseases, but its function in kidney remains unknown. To investigate the role of ANKS6 in cyst formation, we screened an archive of N-ethyl-N-nitrosourea-treated mice and derived a strain carrying a missense mutation (I747N) within the SAM domain of ANKS6. This mutation is only six amino acids away from the PKD-causing mutation (R823W) in cy/+ rats. Evidence of renal cysts in these mice confirmed the crucial role of the SAM domain of ANKS6 in kidney function. Comparative phenotype analysis in cy/+ rats and our Anks6(I747N) mice further showed that the two models display noticeably different PKD phenotypes and that there is a defective interaction between ANKS6 with ANKS3 in the rat and between ANKS6 and BICC1 (bicaudal C homolog 1) in the mouse. Thus, our data demonstrate the importance of ANKS6 for kidney structure integrity and the essential mediating role of its SAM domain in the formation of protein complexes.

Angiotensin II is known to stimulate cardiac hypertrophy and contractility. Most angiotensin II effects are mediated via membrane bound AT1 receptors. However, the role of myocardial AT1 receptors in cardiac hypertrophy and contractility is still rarely defined. To address the hypothesis that increased myocardial AT1 receptor density causes cardiac hypertrophy apart from high blood pressure we developed a transgenic rat model which expresses the human AT1 receptor under the control of the alpha-myosin heavy-chain promoter specifically in the myocardium. Expression was identified and quantified by northern blot analysis and radioligand binding assays, demonstrating overexpression of angiotensin II receptors in the transgenic rats up to 46 times the amount seen in nontransgenic rats. Coupling of the human AT1 receptor to rat G proteins and signal transduction cascade was verified by sensitivity to GTP-gamma-S and increased sensitivity of intracellular Ca2+ [Ca2+]i to angiotensin II in fluo-3 loaded transgenic cardiomyocytes. Transgenic rats exhibited normal cardiac growth and function under baseline conditions. Pronounced hypertrophic growth and contractile responses to angiotensin II, however, were noted in transgenic rats challenged by volume and pressure overload. In summary, we generated a new transgenic rat model that exhibits an upregulated myocardial AT1 receptor density and demonstrates augmented cardiac hypertrophy and contractile response to angiotensin II after volume and pressure overload, but not under baseline conditions.

Abstract —The plasma membrane calmodulin–dependent calcium ATPase (PMCA) is a calcium-extruding enzyme controlling Ca 2+ homeostasis in nonexcitable cells. However, its function in the myocardium is unclear because of the presence of the Na + /Ca 2+ exchanger. We approached the question of the physiological function of the calcium pump using a transgenic “gain of function” model. Transgenic rat lines carrying the human PMCA 4 cDNA under control of the ventricle-specific myosin light chain-2 promoter were established, and expression in the myocardium was ascertained at the mRNA, protein, and functional levels. In vivo hemodynamic measurements in adult homozygous animals showed no differences in baseline and increased cardiac performance recruited by volume overload compared with controls. No differences between transgenic and control cardiomyocytes were found in patch clamp voltage dependence, activation/inactivation behavior of the L -type Ca 2+ current, or fast [Ca 2+ ] i transients (assessed by the Fura-2 method). To test whether the PMCA might be involved in processes other than beat-to-beat regulation of contraction/relaxation, we compared growth processes of neonatal transgenic and control cardiomyocytes. A 1.6- and 2.3-fold higher synthesis rate of total protein was seen in cells from transgenic animals compared with controls on incubation with 2% FCS for 24 hours and 36 hours, respectively. An effect of similar magnitude was observed using 20 μmol/L phenylephrine. A 1.4-fold– and 2.0-fold–higher protein synthesis peak was seen in PMCA-overexpressing cardiomyocytes after stimulation with isoproterenol for 12 hours and 24 hours, respectively. Because pivotal parts of the α- and β-adrenergic signal transduction pathways recently have been localized to caveolae, we tested the hypothesis that the PMCA might alter the amplitude of α- and β-adrenergic growth signals by virtue of its localization in caveolae. Biochemical as well as immunocytochemical studies suggested that the PMCA in large part was colocalized with caveolin 3 in caveolae of cardiomyocytes. These results indicate that the sarcolemmal Ca 2+ -pump has little relevance for beat-to-beat regulation of contraction/relaxation in adult animals but likely plays a role in regulating myocardial growth, possibly through modulation of caveolar signal transduction.

The ovary is one of the organs in which an intrinsic renin-angiotensin system (RAS) has been described. Among angiotensin II receptors, the angiotensin II receptor subtype 2 (AT2) is believed to play an important role in mediating or modulating a variety of intraovarian processes. Previous studies were mainly based on ligand binding techniques using AT2 receptor-specific antagonists. Despite the recent cloning of the AT2 gene, no information is available about the exact intraovarian distribution of AT2 mRNA expression. Therefore, we analyzed ovaries from sexually mature, untreated rats by nonradioactive in situ hybridzation using an AT2-specific anti-sense RNA probe. Experiments were performed on perfusion-fixed ovaries obtained from different stages of the estrous cycle. As an important finding, strong AT2 mRNA expression could be demonstrated exclusively in follicular granulosa cells. Follicles containing AT2 mRNA-positve granulosa cells were mainly in the advanced tertiary stage of follicular development, already exhibiting features of atresia. In addition, individual collapsed, definitive atretic follicles also showed strong AT2 mRNA expression solely in granulosa cells. In corpora lutea and in other structures of the ovary, no message for the AT2 receptor could be detected under these conditions. These data may contribute to a better understanding of the effects exerted by an intraovarian RAS. (J Histochem Cytochem 46:865-870, 1998)

Angiotensin converting enzyme (ACE) is a zinc metalloprotease anchored in the plasma membrane with a carboxy-terminal hydrophobic domain. In addition, the existence of a soluble form of ACE lacking the transmembrane domain has been reported. We show evidence for the existence of an mRNA specific for this isoform produced by alternative splicing. In human umbilical vein endothelial cells, two ACE mRNAs of different length (4.3 and 3.5 kb) were detected by Northern blot. Ribonuclease protection assays and the sequence of a PCR-amplified cDNA fragment show that the shortened ACE mRNA lacks the exons coding for the transmembrane domain of the protein. As this mRNA could be the source of soluble ACE, plasma ACE activity may be regulated on the level of mRNA processing.

The brain renin-angiotensin system plays a role in both cardiovascular homeostasis and neurosecretory functions. Since the mechanisms of angiotensin (Ang) II formation in the human brain have not been clarified, the aims of the present study were to determine the presence of human chymase and angiotensin I-converting enzyme (ACE) in human and non-human brains. In the human brain, the total Ang II-forming activity was significantly higher in the pineal and pituitary glands than those in other regions. In other species (rat, bovine and porcine), the level of chymase as well as total Ang II-forming activities in pineal glands were significantly lower than those in human glands. High levels of chymase-like immunoreactivity (ir) were found in the arteriolar endothelial cells, adventitial mesenchymal cells and in parenchymal cells of the human pineal and pituitary glands while ACE-ir was mostly observed in the endothelial cells and occasionally found in parenchymal cells. Our study provides the first evidence that human chymase exists in the pineal and pituitary glands. The remarkable regional and species differences in mechanisms of Ang II formation suggest a specific role of chymase or ACE in the human brain.

BACKGROUND: Retinal degeneration in transgenic rats that express a mutant cilia gene polycystin-2 (CMV-PKD2(1/703)HA) is characterized by initial photoreceptor degeneration and glial activation, followed by vasoregression and neuronal degeneration (Feng et al., 2009, PLoS One 4: e7328). It is unknown whether glial activation contributes to neurovascular degeneration after photoreceptor degeneration. We characterized the reactivity of Müller glial cells in retinas of rats that express defective polycystin-2. METHODS: Age-matched Sprague-Dawley rats served as control. Retinal slices were immunostained for intermediate filaments, the potassium channel Kir4.1, and aquaporins 1 and 4. The potassium conductance of isolated Müller cells was recorded by whole-cell patch clamping. The osmotic swelling characteristics of Müller cells were determined by superfusion of retinal slices with a hypoosmotic solution. FINDINGS: Müller cells in retinas of transgenic rats displayed upregulation of GFAP and nestin which was not observed in control cells. Whereas aquaporin-1 labeling of photoreceptor cells disappeared along with the degeneration of the cells, aquaporin-1 emerged in glial cells in the inner retina of transgenic rats. Aquaporin-4 was upregulated around degenerating photoreceptor cells. There was an age-dependent redistribution of Kir4.1 in retinas of transgenic rats, with a more even distribution along glial membranes and a downregulation of perivascular Kir4.1. Müller cells of transgenic rats displayed a slight decrease in their Kir conductance as compared to control. Müller cells in retinal tissues from transgenic rats swelled immediately under hypoosmotic stress; this was not observed in control cells. Osmotic swelling was induced by oxidative-nitrosative stress, mitochondrial dysfunction, and inflammatory lipid mediators. INTERPRETATION: Cellular swelling suggests that the rapid water transport through Müller cells in response to osmotic stress is altered as compared to control. The dislocation of Kir4.1 will disturb the retinal potassium and water homeostasis, and osmotic generation of free radicals and inflammatory lipids may contribute to neurovascular injury.

Expression of kidney injury molecule-1 (Kim-1) is rapidly upregulated following tubular injury, constituting a biomarker for acute kidney damage. We examined the renal localization of Kim-1 expression in PKD/Mhm (polycystic kidney disease, Mannheim) (cy/+) rats (cy: mutated allel, +: wild type allel), an established model for autosomal dominant polycystic kidney disease, with chronic, mainly proximal tubulointerstitial alterations. For immunohistochemistry or Western blot analysis, kidneys of male adult heterozygously-affected (cy/+) and unaffected (+/+) littermates were perfusion-fixed or directly removed. Kim-1 expression was determined using peroxidase- or fluorescence-linked immunohistochemistry (alone or in combination with markers for tubule segments or differentiation). Compared to (+/+), only in (cy/+) kidneys, a chronic expression of Kim-1 could be detected by Western blot analysis, which was histologically confined to an apical cellular localization in areas of cystically-transformed proximal tubules with varying size and morphology, but not in distal tubular segments. Kim-1 was expressed by cystic epithelia exhibiting varying extents of dedifferentiation, as shown by double labeling with aquaporin-1, vimentin or osteopontin, yielding partial cellular coexpression. In this model, in contrast to other known molecules indicating renal injury and/or repair mechanisms, the chronic renal expression of Kim-1 is strictly confined to proximal cysts. Its exact role in interfering with tubulo-interstitial alterations in polycystic kidney disease warrants future investigations.

Diabetic retinopathy shares important features with neurodegenerative retinal diseases, including loss of ganglion cells and retinal thinning. The impact on vasoregression and subsequent ischemia-driven changes such as macular edema and proliferative retinopathy are not established. Studies using adult neurodegenerative animal models such as the transgenic TGR(CMV-PKD2(1/703)HA) rat imply early activation of the innate immunity system and the complement system as well as microglia playing a role in the damage of the retinal neurovascular unit.

This study aimed to elucidate the role of the AT2 receptor (AT2R), which is expressed and upregulated in the adrenal zona glomerulosa (ZG) under conditions of increased aldosterone production. We developed a novel transgenic rat (TGR; TGRCXmAT2R) that overexpresses the AT2R in the adrenal gland, heart, kidney, brain, skeletal muscle, testes, lung, spleen, aorta, and vein. As a consequence the total angiotensin II (Ang II) binding sites increased 7.8-fold in the kidney, 25-fold in the heart, and twofold in the adrenals. The AT2R number amounted to 82–98% of total Ang II binding sites. In the ZG of TGRCXmAT2R, the AT2R density was elevated threefold relative to wild-type (WT) littermates, whereas AT1R density remained unchanged. TGRCXmAT2R rats were viable and exhibited normal reproduction, blood pressure, and kidney function. Notably, a slightly but significantly reduced body weight and a moderate increase in plasma urea were observed. With respect to adrenal function, 24-h urinary and plasma aldosterone concentrations were unaffected in TGRCXmAT2R at baseline. Three and 14 days of Ang II infusion (300 ng·min−1·kg−1) increased plasma aldosterone levels in WT and in TGR. These changes were completely abolished by the AT1R blocker losartan. Of note, glomerulosa cell proliferation, as indicated by the number of Ki-67-positive glomerulosa cells, was stimulated by Ang II in TGR and WT rats; however, this increase was significantly attenuated in TGR overexpressing the AT2R. In conclusion, AT2R in the adrenal ZG inhibits Ang II-induced cell proliferation but has no obvious lasting effect on the regulation of the aldosterone production at the investigated stages.

Several intervention studies with angiotensin I converting enzyme (ACE) inhibitors have demonstrated a remarkable improvement in the treatment of patients with primary hypertension and congestive heart failure. Since ACE inhibitor therapy in patients with congestive heart failure not only improves systemic haemodynamics but also provides a better prognosis, the cardiac renin-angiotensin system is apparently one of the major targets of ACE inhibitor therapy. Recent studies provided evidence that the human heart contains high affinity Ang II (Ang II) receptors with both subtype population and ACE. In addition to ACE, a novel cardiac Ang II forming enzyme (human chymase) has been identified in human hearts. Unlike in the rat heart, the minor (10%) component of Ang II-forming activity in the left ventricle is due to ACE, whereas the major (80%) component is due to human chymase. This novel cardiac serine proteinase has been purified from the human left ventricle and characterized, and recently, the cDNA and the gene for this enzyme have been cloned. Biochemical characterization revealed that human chymase is the most efficient and specific Ang II-forming enzyme described thus far, but the cellular and regional distribution of the two Ang II-forming enzymes seems to be different. ACE is mainly localized in endothelial cells and fibroblasts and the expression level is higher in atria than ventricles, whereas chymase is synthesized and stored in secretory granules of mast cells, endothelial cells, and mesenchymal cells, and after its secretion localized in the interstitial region of the myocardium and its expression is higher in ventricles than atria. These results imply distinct roles of these two Ang II-forming enzymes in cardiac Ang II formation and in the physiological function of the human heart. Since localization of cardiac renin and angiotensinogen were also identified in human heart, it is important to understand the detailed mechanisms of the tissue Ang II formation and its contribution to the pathophysiological changes in cardiovascular diseases.

Primary hypertension is a disease with a significant genetic background. Although we do not know exactly which genes are involved, present studies link sections of the genome to the hypertensive phenotype in genetically hypertensive rats (SHRsp). In addition, several candidate genes have been proposed to be related to this disorder. They include components of vaso­ active regulatory systems, long considered to be involved in the pathogenesis of high blood pressure. The capability to modify the expression of specific gene products in intact animals provides a powerful experimental approach to study cardiovascular pathophysiology. Such animal models can be generated using transgenic technology. Transgenic animals are useful for the production of defined alterations in the germ line that result in specific pathophysiologic changes. These changes can be induced not only in a systemic but also in a tissue-specific manner by studying the functional aspects of organor cell

Vasoregression is a hallmark of vascular eye diseases but the mechanisms involved are still largely unknown. We have recently characterized a rat ciliopathy model which develops primary photoreceptor degeneration and secondary vasoregression. To improve the understanding of secondary vasoregression in retinal neurodegeneration, we used microarray techniques to compare gene expression profiles in this new model before and after retinal vasoregression. Differential gene expression was validated by quantitative RT-PCR, Western blot and immunofluorescence. Of the 157 genes regulated more than twofold, the MHC class II invariant chain CD74 yielded the strongest upregulation, and was allocated to activated microglial cells close to the vessels undergoing vasoregression. Pathway clustering identified genes of the immune system including inflammatory signaling, and components of the complement cascade upregulated during vasoregression. Together, our data suggest that microglial cells involved in retinal immune response participate in the initiation of vasoregression in the retina.

Background Neuronal damage is correlated with vascular dysfunction in the diseased retina, but the underlying mechanisms remain controversial because of the lack of suitable models in which vasoregression related to neuronal damage initiates in the mature retinal vasculature. The aim of this study was to assess the temporal link between neuronal damage and vascular patency in a transgenic rat (TGR) with overexpression of a mutant cilia gene polycystin-2. Methods Vasoregression, neuroglial changes and expression of neurotrophic factors were assessed in TGR and control rats in a time course. Determination of neuronal changes was performed by quantitative morphometry of paraffin-embedded vertical sections. Vascular cell composition and patency were assessed by quantitative retinal morphometry of digest preparations. Glial activation was assessed by western blot and immunofluorescence. Expression of neurotrophic factors was detected by quantitative PCR. Findings At one month, number and thickness of the outer nuclear cell layers (ONL) in TGR rats were reduced by 31% (p

SummaryAngiopoietin-2 (Ang-2) antagonises the maturing effect of angiopoietin-1 (Ang-1) on blood vessels, and cooperates with VEGF to induce neovascularisation. In knockout mice,Ang-2 displayed a specific role in postnatal angiogenic remodelling. Here, we demonstrate that mice deficient in Ang-2 fail to form a proper spatial retinal vascular network. The retinal vasculature was characterised by reduced large vessel numbers and defects forming the superficial periphery mostly on the arteriolar site, and the secondary and tertiary deep capillary network. Hypoxia in the retinal periphery induced a four-fold VEGF upregulation and active endothelial proliferation for up to 60 days. Concomitantly, retinal digest preparations showed increased arteriolar (+33%) and capillary diameters (+90%), and fluorescein angiograms revealed leakiness of neovascular front. At one year of age, persistent preretinal vessels were non-leaky in accordance with a relative increase in the ratio of Ang-1 to VEGF.Taken together, the data suggest that Ang-2 has an important function in the spatial configuration of the three-dimensional retinal vasculature. Secondarily, prolonged VEGF activity results in a model of persistent proliferative retinopathy.

OBJECTIVE— The mechanism underlying pericyte loss during incipient diabetic retinopathy remains controversial. Hyperglycemia induces angiopoietin-2 (Ang-2) transcription, which modulates capillary pericyte coverage. In this study, we assessed loss of pericyte subgroups and the contribution of Ang-2 to pericyte migration. RESEARCH DESIGN AND METHODS— Numbers of total pericytes and their subgroups were quantified in retinal digest preparations of spontaneous diabetic XLacZ mice. Pericytes were divided into subgroups according to their localization, their position relative to adjacent endothelial cells, and the expression of LacZ. The contribution of Ang-2 to pericyte migration was assessed in Ang-2 overexpressing (mOpsinhAng2) and deficient (Ang2LacZ) mice. RESULTS— Pericyte numbers were reduced by 16% (P < 0.01) in XLacZ mice after 6 months of diabetes. Reduction of pericytes was restricted to pericytes on straight capillaries (relative reduction 27%, P < 0.05) and was predominantly observed in LacZ-positive pericytes (−20%, P < 0.01). Hyperglycemia increased the numbers of migrating pericytes (69%; P < 0.05), of which the relative increase due to diabetes was exclusively in LacZ-negative pericytes, indicating reduced adherence to the capillaries (176%; P < 0.01). Overexpression of Ang-2 in nondiabetic retinas mimicked diabetic pericyte migration of wild-type animals (78%; P < 0.01). Ang-2 deficient mice completely lacked hyperglycemia-induced increase in pericyte migration compared with wild-type littermates. CONCLUSIONS— Diabetic pericyte loss is the result of pericyte migration, and this process is modulated by the Ang-Tie system.

Retinae of aged humans show signs of vascular regression. Vascular regression involves a mismatch between Angiopoietin-2 (Ang-2) and vascular endothelial growth factor (VEGF) expression. We used heterozygous Ang-2 deficient (Ang2LacZ) mice to evaluate murine retinal vascular changes and gene expression of growth factors. Vascular changes were assessed by quantitative retinal morphometry and gene expression levels of growth factors were measured by quantitative PCR. The numbers of endothelial cells and pericytes did not change in the Ang2LacZ retinae with age, whereas they decreased throughout the age spectrum studied in the wild type retinae. Moreover, vascular regression significantly decelerated in the heterozygous Ang2LacZ retinae (200% to 1 month), while the formation of acellular capillaries was significantly increased at 13 months in the wild type retinae (340% to 1 month). Gene expression analysis revealed that VEGF, Ang-1, PDGF-B and Ang2 mRNA levels were decreased in the wild type retinae at 9 month of age. However, the decrease of Ang-2 was smaller compared with other genes. While VEGF levels dropped in wild type mice up to 60% compared to 1 month, VEGF increased in heterozygous Ang-2 deficient retinae at an age of 9 months (141% to 1 month). Similarly, Ang-1 levels decreased in wild type mice (45% to 1 month), but remained stable in Ang2LacZ mice. These data suggest that Ang-2 gene dose reduction decelerates vasoregression in the retina with age. This effect links to higher levels of survival factors such as VEGF and Ang-1, suggesting that the ratio of these factors is critical for capillary cell survival.