Sigrid Hoffmann

Pericyte Migration : A Novel Mechanism of Pericyte Loss in Experimental Diabetic 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.

Mechanisms of angiotensin II signaling on cytoskeleton of podocytes

Podocytes are significant in establishing the glomerular filtration barrier. Sustained rennin–angiotensin system (RAS) activation is crucial in the pathogenesis of podocyte injury and causes proteinuria. This study demonstrates that angiotensin II (Ang II) caused a reactive oxygen species (ROS)-dependent rearrangement of cortical F-actin and a migratory phenotype switch in cultured mouse podocytes with stable Ang II type 1 receptor (AT1R) expression. Activated small GTPase Rac-1 and phosphorylated ezrin/radixin/moesin (ERM) proteins provoked Ang II-induced F-actin cytoskeletal remodeling. This work also shows increased expression of Rac-1 and phosphorylated ERM proteins in cultured podocytes, and in glomeruli of podocyte-specific AT1R transgenic rats (Neph-hAT1 TGRs). The free radical scavenger DMTU eliminated Ang II-induced cell migration, ERM protein phosphorylation and cortical F-actin remodeling, indicating that ROS mediates the influence of Rac-1 on podocyte AT1R signaling. Heparin, a potent G-coupled protein kinase 2 inhibitor, was found to abolish ERM protein phosphorylation and cortical F-actin ring formation in Ang II-treated podocytes, indicating that phosphorylated ERM proteins are the cytoskeletal effector in AT1R signaling. Moreover, Ang II stimulation triggered down-regulation of α actinin-4 and reduced focal adhesion expression in podocytes. Signaling inhibitor assay of Ang II-treated podocytes reveals that Rac-1, RhoA, and F-actin reorganization were involved in expressional regulation of α actinin-4 in AT1R signaling. With persistent RAS activation, the Ang II-induced phenotype shifts from being dynamically stable to adaptively migratory, which may eventually exhaust podocytes with a high actin cytoskeletal turnover, causing podocyte depletion and focal segmental glomerulosclerosis.

Impaired pericyte recruitment and abnormal retinal angiogenesis as a result of angiopoietin-2 overexpression

Angiopoietin-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 < 0.01) and a 46% increase of vascular density of the capillary network at postnatal day 10 compared to wild type mice. In the model of oxygen-induced retinopathy (OIR), Ang2 overexpression resulted in enhanced preretinal (+103%) and intraretinal neovascularization (+29%). Newly formed intraretinal vessels in OIR were also pericyte-deficient (-26%; p < 0.01). The total expression of Ang2 in transgenic mice was seven-fold, compared with wild type controls. Ang2 modulated expression of genes encoding VEGF (+65%) and Ang1 (+79%) in transgenic animals. These data suggest that Ang2 is involved in pericyte recruitment, and modulates intraretinal, and preretinal vessel formation in the eye under physiological and pathological conditions.

Both Lymphotoxin-α and TNF Are Crucial for Control of Toxoplasma gondii in the Central Nervous System

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-α (LTα), is still unknown. Upon oral infection with T. gondii, TNF−/−, LTα−/−, and TNF/LTα−/− 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−/−, LTα−/−, and TNF/LTα−/− mice as compared with WT animals. Frequencies of parasite-specific IFN-γ-producing T cells, intracerebral and splenic IFN-γ production, and T. gondii-specific IgM and IgG titers in LTα−/− and TNF/LTα−/− 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−/−, LTα−/−, and TNF/LTα−/−, 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-γ-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 LTα for survival of toxoplasmosis. These findings demonstrate the crucial role of both LTα and TNF for control of intracerebral toxoplasms.

Missense mutation in sterile alpha motif of novel protein SamCystin is associated with polycystic kidney disease in (cy/+)rat

Autosomal dominant polycystic kidney disease (PKD) is the most common genetic disease that leads to kidney failure in humans. In addition to the known causative genes PKD1 and PKD2, there are mutations that result in cystic changes in the kidney, such as nephronophthisis, autosomal recessive polycystic kidney disease, or medullary cystic kidney disease. Recent efforts to improve the understanding of renal cystogenesis have been greatly enhanced by studies in rodent models of PKD. Genetic studies in the (cy/+) rat showed that PKD spontaneously develops as a consequence of a mutation in a gene different from the rat orthologs of PKD1 and PKD2 or other genes that are known to be involved in human cystic kidney diseases. This article reports the positional cloning and mutation analysis of the rat PKD gene, which revealed a C to T transition that replaces an arginine by a tryptophan at amino acid 823 in the protein sequence. It was determined that Pkdr1 is specifically expressed in renal proximal tubules and encodes a novel protein, SamCystin, that contains ankyrin repeats and a sterile alpha motif. The characterization of this protein, which does not share structural homologies with known polycystins, may give new insights into the pathophysiology of renal cyst development in patients.

MuRF1-dependent Regulation of Systemic Carbohydrate Metabolism as Revealed from Transgenic Mouse Studies

Under various pathophysiological muscle-wasting conditions, such as diabetes and starvation, a family of ubiquitin ligases, including muscle-specific RING-finger protein 1 (MuRF1), are induced to target muscle proteins for degradation via ubiquitination. We have generated transgenic mouse lines over-expressing MuRF1 in a skeletal muscle-specific fashion (MuRF1-TG mice) in an attempt to identify the in vivo targets of MuRF1. MuRF1-TG lines were viable, had normal fertility and normal muscle weights at eight weeks of age. Comparison of quadriceps from MuRF1-TG and wild type mice did not reveal elevated multi-ubiquitination of myosin as observed in human patients with muscle wasting. Instead, MuRF1-TG mice expressed lower levels of pyruvate dehydrogenase (PDH), a mitochondrial key enzyme in charge of glycolysis, and of its regulator PDK2. Furthermore, yeast two-hybrid interaction studies demonstrated the interaction of MuRF1 with PDH, PDK2, PDK4, PKM2 (all participating in glycolysis) and with phosphorylase beta (PYGM) and glycogenin (both regulating glycogen metabolism). Consistent with the idea that MuRF1 may regulate carbohydrate metabolism, MuRF1-TG mice had twofold elevated insulin blood levels and lower hepatic glycogen contents. To further examine MuRF1s role for systemic carbohydrate regulation, we performed glucose tolerance tests (GTT) in wild type and MuRF1-TG mice. During GTT, MuRF1-TG mice developed striking hyperinsulinaemia and hepatic glycogen stores, that were depleted at basal levels, became rapidly replenished. Taken together, our data demonstrate that MuRF1 expression in skeletal muscle re-directs glycogen synthesis to the liver and stimulates pancreatic insulin secretion, thereby providing a regulatory feedback loop that connects skeletal muscle metabolism with the liver and the pancreas during metabolic stress.

Vasoregression Linked to Neuronal Damage in the Rat with Defect of Polycystin-2

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<0.001) and 17% (p<0.05), respectively, compared to age-matched control rats. Furthermore, the reduction progressed from 1 to 7 months in TGR rats. Apoptosis was selectively detected in the photoreceptor in the ONL, starting after one month. Nevertheless, TGR and control rats showed normal responses in electroretinogram at one month. From the second month onwards, TGR retinas had significantly increased acellular capillaries (p<0.001), and a reduction of endothelial cells (p<0.01) and pericytes (p<0.01). Upregulation of GFAP was first detected in TGR retinas after 1 month in glial cells, in parallel with an increase of FGF2 (fourfold) and CNTF (60 %), followed by upregulation of NGF (40 %) at 3 months. Interpretation Our data suggest that TGR is an appropriate animal model for vasoregression related to neuronal damage. Similarities to experimental diabetic retinopathy render this model suitable to understand general mechanisms of maturity-onset vasoregression.

Endothelial survival factors and spatial completion, but not pericyte coverage of retinal capillaries determine vessel plasticity

Pericyte loss and capillary regression are characteristic for incipient diabetic retinopathy. Pericyte recruitment is involved in vessel maturation, and ligand‐receptor systems contributing to pericyte recruitment are survival factors for endothelial cells in pericyte‐free in vitro systems. We studied pericyte recruitment in relation to the susceptibility toward hyperoxia‐induced vascular remodeling using the pericyte reporter X‐LacZ mouse and the mouse model of retinopathy of prematurity (ROP). Pericytes were found in close proximity to vessels, both during formation of the superficial and the deep capillary layers. When exposure of mice to the ROP was delayed by 24 h, i.e., after the deep retinal layer had formed [at postnatal (p) day 8], preretinal neovascularizations were substantially diminished at p18. Mice with a delayed ROP exposure had 50% reduced avascular zones. Formation of the deep capillary layers at p8 was associated with a combined up‐regulation of angiopoietin‐1 and PDGF‐B, while VEGF was almost unchanged during the transition from a susceptible to a resistant capillary network. Inhibition of Tie‐2 function either by soluble Tie‐2 or by a sulindac analog, an inhibitor of Tie‐2 phosphorylation, resensitized retinal vessels to neovascularizations due to a reduction of the deep capillary network. Inhibition of Tie‐2 function had no effect on pericyte recruitment. Our data indicate that the final maturation of the retinal vasculature and its resistance to regressive signals such as hyperoxia depend on the completion of the multilayer structure, in particular the deep capillary layers, and are independent of the coverage by pericytes.

A renin transcript lacking exon 1 encodes for a non-secretory intracellular renin that increases aldosterone production in transgenic rats.

Renin transcripts lacking exon 1 and thus the signal sequence for co‐translational transport to the endoplasmatic reticulum encode for a protein (exon[2‐9]renin), that is confined to the cytoplasm. The function of exon(2‐9)renin is currently unknown. Mitochondrial renin increases under conditions which stimulate aldosterone production. We hypothesized that exon(2‐9)renin (1) is translated into a functionally active protein in vivo, (2) is not secreted but remains within the cytoplasm and (3) stimulates aldosterone production. To test these hypotheses we generated transgenic rats overexpressing exon(2‐9)renin. Four transgenic lines were obtained expressing the transcript in various tissues including the heart and the adrenal gland. Renin was enriched particularly in the cytoplasm of transgenic rats. Renin was not elevated in plasma, indicating that exon(2‐9)renin is produced but not secreted. The ratio of aldosterone to renin concentrations in plasma (PAC/PRC) was elevated in all transgenic lines except line 307, which also did not exhibit elevated cytoplasmatic renin levels in the adrenal gland (PAC/PRC in controls: 2.8±2.3; line 307: 1.9±0.8; n. s.; line 284: 5.8±1.9; P<0.02; line 294: 5.0±2.3; P<0.001; line 276: 10.3±5.1; P<0.001). We conclude that the exon(1A‐9) renin transcript (1) is translated into a functionally active intracellular protein; (2) is targeted to the cytoplasm rather than being sorted to the secretory pathways and (3) is functionally active, regulating aldosterone production. The CX‐(exon2‐9)renin transgenic rat appears to be a useful model to study the role and the mechanisms of action of cytoplasmatic renin derived from exon(1A‐9) transcripts.

Downregulation of the antioxidant protein peroxiredoxin 2 contributes to angiotensin II–mediated podocyte apoptosis

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.