Reduction of cortical parvalbumin-expressing GABAergic interneurons in a rodent hyperoxia model of preterm birth brain injury with deficits in social behavior and cognition.

The inhibitory GABAergic system in the brain is involved in the etiology of various psychiatric problems, including autism spectrum disorders (ASD), attention deficit hyperactivity disorder (ADHD) and others. These disorders are influenced not only by genetic but also by environmental factors, such as preterm birth, although the underlying mechanisms are not known. In a translational hyperoxia model, exposing mice pups at P5 to 80% oxygen for 48 h to mimic a steep rise of oxygen exposure caused by preterm birth from in utero into room air, we documented a persistent reduction of cortical mature parvalbumin-expressing interneurons until adulthood. Developmental delay of cortical myelin was observed, together with decreased expression of oligodendroglial glial cell-derived neurotrophic factor (GDNF), a factor involved in interneuronal development. Electrophysiological and morphological properties of remaining interneurons were unaffected. Behavioral deficits were observed for social interaction, learning and attention. These results demonstrate that neonatal oxidative stress can lead to decreased interneuron density and to psychiatric symptoms. The obtained cortical myelin deficit and decreased oligodendroglial GDNF expression indicate that an impaired oligodendroglial-interneuronal interplay contributes to interneuronal damage.

The dipeptidyl peptidase inhibitor linagliptin and the angiotensin II receptor blocker telmisartan show renal benefit by different pathways in rats with 5/6 nephrectomy.

Dipeptidyl peptidase (DPP)-4 inhibitors delay chronic kidney disease (CKD) progression in experimental diabetic nephropathy in a glucose-independent manner. Here we compared the effects of the DPP-4 inhibitor linagliptin versus telmisartan in preventing CKD progression in non-diabetic rats with 5/6 nephrectomy. Animals were allocated to 1 of 4 groups: sham operated plus placebo; 5/6 nephrectomy plus placebo; 5/6 nephrectomy plus linagliptin; and 5/6 nephrectomy plus telmisartan. Interstitial fibrosis was significantly decreased by 48% with linagliptin but a non-significant 24% with telmisartan versus placebo. The urine albumin-to-creatinine ratio was significantly decreased by 66% with linagliptin and 92% with telmisartan versus placebo. Blood pressure was significantly lowered by telmisartan, but it was not affected by linagliptin. As shown by mass spectrometry, the number of altered peptide signals for linagliptin in plasma was 552 and 320 in the kidney. For telmisartan, there were 108 peptide changes in plasma and 363 in the kidney versus placebo. Linagliptin up-regulated peptides derived from collagen type I, apolipoprotein C1, and heterogeneous nuclear ribonucleoproteins A2/B1, a potential downstream target of atrial natriuretic peptide, whereas telmisartan up-regulated angiotensin II. A second study was conducted to confirm these findings in 5/6 nephrectomy wild-type and genetically deficient DPP-4 rats treated with linagliptin or placebo. Linagliptin therapy in wild-type rats was as effective as DPP-4 genetic deficiency in terms of albuminuria reduction. Thus, linagliptin showed comparable efficacy to telmisartan in preventing CKD progression in non-diabetic rats with 5/6 nephrectomy. However, the underlying pathways seem to be different.

DPP-4 inhibition on top of angiotensin receptor blockade offers a new therapeutic approach for diabetic nephropathy.

BACKGROUND: The need for an improved treatment for diabetic nephropathy is greatest in patients who do not adequately respond to angiotensin II receptor blockers (ARBs). This study investigated the effect of the novel dipeptidyl peptidase-4 inhibitor linagliptin alone and in combination with the ARB telmisartan on the progression of diabetic nephropathy in diabetic endothelial nitric oxide synthase (eNOS) knockout mice. METHODS: Sixty male eNOS knockout C57BL/6J mice were divided into four groups after receiving intraperitoneal highdose streptozotocin: telmisartan (1 mg/kg), linagliptin (3 mg/kg), linagliptin + telmisartan (3 mg/kg + 1 mg/kg) and vehicle. Fourteen mice were used as non-diabetic controls. RESULTS: After 12 weeks, urine and blood were obtained and blood pressure measured. Glucose concentrations were increased and similar in all diabetic groups. Telmisartan alone reduced systolic blood pressure by 5.9 mmHg versus diabetic controls (111.2 ± 2.3 mmHg vs 117.1 ± 2.2 mmHg; mean ± SEM; P=0.071). Combined treatment significantly reduced albuminuria compared with diabetic controls (71.7 ± 15.3 µg/24 h vs. 170.8 ± 34.2 µg/24 h; P=0.017), whereas the effects of single treatment with either telmisartan (97.8 ± 26.4 µg/24 h) or linagliptin (120.8 ± 37.7 µg/24 h) were not statistically significant. DPP-4 inhibition, alone and in combination, led to significantly lower plasma osteopontin levels compared with telmisartan alone. Histological analysis revealed reduced glomerulosclerosis after Linagliptin alone and in combination with telmisartan in comparison to non treated diabetic animals (p<0.01 and p<0.05). Kidney malonaldehyde immune-reactivity, a marker of oxidative stress, was significantly lower in animals treated with linagliptin. CONCLUSIONS: DPP-4 inhibition on top of ARB treatment significantly reduced urinary albumin excretion and oxidative stress in diabetic eNOS knockout mice. Linagliptin on top of an angiotensin II receptor blocker may offer a new therapeutic approach for patients with diabetic nephropathy.

Renoprotective effects of combined endothelin-converting enzyme/neutral endopeptidase inhibitor SLV338 in acute and chronic experimental renal damage.

BACKGROUND: Acute kidney injury (AKI) as well as chronic renal failure are associated with a huge mortality/morbidity. However, so far no drugs have been approved for the treatment of acute kidney failure and only a few for the treatment of chronic kidney disease (CKD). We analysed the effect of SLV338, a neutral endopeptidase (NEP)/endothelin converting enzyme (ECE)-inhibitor in animal models of acute kidney failure as well as chronic renal failure. METHODS: Acute renal failure was induced in male Wistar rats by uninephrectomy and clamping of the remaining kidney for 55 minutes. SLV338 (total dose: 4.9 mg/kg) or vehicle was continuously infused for 2 hours (starting 20 minutes prior to clamping). Sham operated animals served as controls. Plasma creatinine was measured at baseline and day 2 and 8 after renal ischemia-reperfusion. Hypertensive renal damage was induced in male Sprague Dawley rats by nitric oxide deficiency using L-NAME (50 mg/kg per day, added to drinking water for 4 weeks). One group was treated over the same time period with SLV338 (30 mg/kg per day, mixed with food). Systolic blood pressure was monitored weekly. At study end, urine and blood samples were collected and kidneys were harvested. RESULTS: Acute renal ischemia-reperfusion caused a 5-fold plasma creatinine elevation (day 2), which was significantly attenuated by more than 50% in animals treated with SLV338 (p < 0.05). Renal failure was accompanied by a 67% mortality in vehicle-treated rats, but only 20% after SLV338 treatment (p = 0.03 compared to sham controls). Chronic L-NAME administration caused hypertension, urinary albumin excretion, glomerulosclerosis, renal arterial remodelling, and renal interstitial fibrosis. Treatment with SLV338 did not significantly affect blood pressure, but abolished renal tissue damage (interstitial fibrosis, glomerulosclerosis, renal arterial remodelling (p < 0.05 versus L-NAME group in each case). CONCLUSIONS: The dual ECE/NEP inhibitor SLV338 preserves kidney function and reduces mortality in severe acute ischemic renal failure. Moreover, combined ECE/NEP inhibition prevents hypertensive renal tissue damage in a blood pressure independent manner in L-NAME-treated rats.

Neonatal Hyperoxia Perturbs Neuronal Development in the Cerebellum.

Impaired postnatal brain development of preterm infants often results in neurological deficits. Besides pathologies of the forebrain, maldeveolopment of the cerebellum is increasingly recognized to contribute to psychomotor impairments of many former preterm infants. However, causes are poorly defined. We used a hyperoxia model to define neonatal damage in cerebellar granule cell precursors (GCPs) and in Purkinje cells (PCs) known to be essential for interaction with GCPs during development. We exposed newborn rats to 24 h 80% O2 from age P6 to P7 to identify postnatal and long-term damage in cerebellar GCPs at age P7 after hyperoxia and also after recovery in room air thereafter until P11 and P30. We determined proliferation and apoptosis of GCPs and immature neurons by immunohistochemistry, quantified neuronal damage by qPCR and Western blots for neuronal markers, and measured dendrite outgrowth of PCs by CALB1 immunostainings and by Sholl analysis of Golgi stainings. After hyperoxia, proliferation of PAX6+ GCPs was decreased at P7, while DCX + CASP3+ cells were increased at P11. Neuronal markers Pax6, Tbr2, and Prox1 were downregulated at P11 and P30. Neuronal damage was confirmed by reduced NeuN protein expression at P30. Sonic hedgehog (SHH) was significantly decreased at P7 and P11 after hyperoxia and coincided with lower CyclinD2 and Hes1 expression at P7. The granule cell injury was accompanied by hampered PC maturation with delayed dendrite formation and impaired branching. Neonatal injury induced by hyperoxia inhibits PC functioning and impairs granule cell development. As a conclusion, maldevelopment of the cerebellar neurons found in preterm infants could be caused by postnatal oxygen toxicity.

Blood pressure and glucose independent renoprotective effects of dipeptidyl peptidase-4 inhibition in a mouse model of type-2 diabetic nephropathy.

BACKGROUND: Despite the beneficial effects of type 4 dipeptidyl peptidase (DPP-4) inhibitors on glucose levels, its effects on diabetic nephropathy remain unclear. METHOD: This study examined the long-term renoprotective effects of DPP-4 inhibitor linagliptin in db/db mice, a model of type 2 diabetes. Results were compared with the known beneficial effects of renin-angiotensin system blockade by enalapril. Ten-week-old male diabetic db/db mice were treated for 3 months with either vehicle (n = 10), 3 mg linagliptin/kg per day (n = 8), or 20 mg enalapril/kg per day (n = 10). Heterozygous db/m mice treated with vehicle served as healthy controls (n = 8). RESULTS: Neither linagliptin nor enalapril had significant effects on the parameters of glucose metabolism or blood pressure in diabetic db/db mice. However, linagliptin treatment reduced albuminuria and attenuated kidney injury. In addition, expression of podocyte marker podocalyxin was normalized. We also analysed DPP-4 expression by immunofluorescence in human kidney biopsies and detected upregulation of DPP-4 in the glomeruli of patients with diabetic nephropathy, suggesting that our findings might be of relevance for human kidney disease as well. CONCLUSION: Treatment with DPP-4 inhibitor linagliptin delays the progression of diabetic nephropathy damage in a glucose-independent and blood-pressure-independent manner. The observed effects may be because of the attenuation of podocyte injury and inhibition of myofibroblast transformation.

Angiotensin type 2 receptor stimulation ameliorates left ventricular fibrosis and dysfunction via regulation of tissue inhibitor of matrix metalloproteinase 1/matrix metalloproteinase 9 axis and transforming growth factor ß1 in the rat heart.

Left ventricular (LV) remodeling is the main reason for the development of progressive cardiac dysfunction after myocardial infarction (MI). This study investigated whether stimulation of the angiotensin type 2 receptor is able to ameliorate post-MI cardiac remodeling and what the underlying mechanisms may be. MI was induced in Wistar rats by permanent ligation of the left coronary artery. Treatment with the angiotensin type 2 receptor agonist compound 21 (0.03 mg/kg) was started 6 hours post-MI and continued for 6 weeks. Hemodynamic parameters were measured by echocardiography and intracardiac catheter. Effects on proteolysis were studied in heart tissue and primary cardiac fibroblasts. Compound 21 significantly improved systolic and diastolic functions, resulting in improved ejection fraction (71.2±4.7% versus 53.4±7.0%; P<0.001), fractional shortening (P<0.05), LV internal dimension in systole (P<0.05), LV end-diastolic pressure (16.9±1.2 versus 22.1±1.4 mm Hg; P<0.05), ratio of early (E) to late (A) ventricular filling velocities, and maximum and minimum rate of LV pressure rise (P<0.05). Compound 21 improved arterial stiffness parameters and reduced collagen content in peri-infarct myocardium. Tissue inhibitor of matrix metalloproteinase 1 was strongly upregulated, whereas matrix metalloproteinases 2 and 9 and transforming growth factor ß1 were diminished in LV of treated animals. In cardiac fibroblasts, compound 21 initially induced tissue inhibitor of matrix metalloproteinase 1 expression followed by attenuated matrix metalloproteinase 9 and transforming growth factor ß1 secretion. In conclusion, angiotensin type 2 receptor stimulation improves cardiac function and prevents cardiac remodeling in the late stage after MI, suggesting that angiotensin type 2 receptor agonists may be considered a future pharmacological approach for the improvement of post-MI cardiac dysfunction.

The novel DPP-4 inhibitors linagliptin and BI 14361 reduce infarct size after myocardial ischemia/reperfusion in rats.

BACKGROUND: Dipeptidylpeptidase-4 inhibition is reported to have beneficial effects on myocardial ischemia. Mechanisms might include a reduced degradation of stromal cell-derived factor-1 alpha with subsequent increased recruitment of circulating stem cells and/or incretin receptor-dependent pathways. This study evaluated the novel xanthine-based dipeptidylpeptidase-4 inhibitors linagliptin (BI 1356) and BI 14361 in cardiac ischemia. METHODS: Male Wistar rats were pretreated with linagliptin or BI 14361 and subjected to ligation of the left anterior descending coronary artery for 30 min. RESULTS: Dipeptidylpeptidase-4 inhibition significantly reduced the infarct size after 7 days (-27.7%, p<0.05) and 8 weeks (-18.0%, p<0.05). There was a significantly improved maximum rate of left ventricular pressure decline (dP/dt min) in linagliptin-treated animals 8 weeks after ischemia/reperfusion. Apart from that, treatment did not improve cardiac function as determined by echocardiography and cardiac catheterization. Immunohistological staining revealed an increased number of cells positive for stromal cell-derived factor-1 alpha, CXCR-4 and CD34 within and around the infarcted area of BI 14361-treated animals. CONCLUSIONS: Linagliptin and BI 14361 are able to reduce infarct size after myocardial ischemia. The immunohistological findings support the hypothesis that dipeptidylpeptidase-4 inhibition via reduced cleavage of stromal cell-derived factor-1 alpha might lead to an enhanced recruitment of CXCR-4+ circulating progenitor cells.

Effects of stimulation of soluble guanylate cyclase on diabetic nephropathy in diabetic eNOS knockout mice on top of angiotensin II receptor blockade.

The prevalence of diabetes mellitus and its complications, such as diabetic nephropathy (DN), is rising worldwide and prevention and treatment are therefore becoming increasingly important. Therapy of DN is particularly important for patients who do not adequately respond to angiotensin receptor blocker (ARB) treatment. Novel approaches include the stimulation of soluble guanylate cyclase (sGC) as it is reported to have beneficial effects on cardiac and renal damage. We aimed to investigate the effects of the sGC stimulator riociguat and ARB telmisartan on kidney function and structure in a hypertensive model of diabetic nephropathy. Seventy-six diabetic male eNOS knockout C57BL/6J mice were randomly divided after having received streptozotocin: telmisartan (1 mg/kg/d), riociguat (3 mg/kg/d), riociguat+telmisartan (3+1 mg/kg/d), and vehicle. Fourteen mice were used as non-diabetic controls. Treatment duration was 11 weeks. Glucose concentrations were increased and similar in all diabetic groups. Telmisartan insignificantly reduced blood pressure by 5.9 mmHg compared with diabetic controls (111.2±2.3 mmHg vs. 117.1±2.2 mmHg; p = 0.071). Treatment with riociguat both alone and in combination with telmisartan led to a significant reduction of blood pressure towards diabetic vehicle (105.2±2.5 mmHg and 105.0±3.2 mmHg, respectively, vs. 117.1±2.2 mmHg). Combined treatment also significantly decreased albuminuria compared with diabetic controls (47.3±9.6 µg/24 h vs. 170.8±34.2 µg/24 h; p = 0.002) reaching levels similar to those of non-diabetic controls (34.4±10.6 µg/24 h), whereas the reduction by single treatment with either telmisartan (97.8±26.4 µg/24 h) or riociguat (97.1±15.7 µg/24 h) was not statistically significant. The combination treatment led to a significant (p<0.01) decrease of tissue immunoreactivity of malondialdehyde, as consequence of reduced oxidative stress. In conclusion, stimulation of sGC significantly reduced urinary albumin excretion in diabetic eNOS knockout mice treated already with ARB. Thus, this new drug class on top of standard ARBs administration may offer a new therapeutic approach for patients resistant to ARB treatment.

Soluble guanylate cyclase stimulation prevents fibrotic tissue remodeling and improves survival in salt-sensitive Dahl rats.

BACKGROUND: A direct pharmacological stimulation of soluble guanylate cyclase (sGC) is an emerging therapeutic approach to the management of various cardiovascular disorders associated with endothelial dysfunction. Novel sGC stimulators, including riociguat (BAY 63-2521), have a dual mode of action: They sensitize sGC to endogenously produced nitric oxide (NO) and also directly stimulate sGC independently of NO. Little is known about their effects on tissue remodeling and degeneration and survival in experimental malignant hypertension. METHODS AND RESULTS: Mortality, hemodynamics and biomarkers of tissue remodeling and degeneration were assessed in Dahl salt-sensitive rats maintained on a high salt diet and treated with riociguat (3 or 10 mg/kg/d) for 14 weeks. Riociguat markedly attenuated systemic hypertension, improved systolic heart function and increased survival from 33% to 85%. Histological examination of the heart and kidneys revealed that riociguat significantly ameliorated fibrotic tissue remodeling and degeneration. Correspondingly, mRNA expression of the pro-fibrotic biomarkers osteopontin (OPN), tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) and plasminogen activator inhibitor-1 (PAI-1) in the myocardium and the renal cortex was attenuated by riociguat. In addition, riociguat reduced plasma and urinary levels of OPN, TIMP-1, and PAI-1. CONCLUSIONS: Stimulation of sGC by riociguat markedly improves survival and attenuates systemic hypertension and systolic dysfunction, as well as fibrotic tissue remodeling in the myocardium and the renal cortex in a rodent model of pressure and volume overload. These findings suggest a therapeutic potential of sGC stimulators in diseases associated with impaired cardiovascular and renal functions.

Endothelin-converting enzyme/neutral endopeptidase inhibitor SLV338 prevents hypertensive cardiac remodeling in a blood pressure-independent manner.

Hypertensive heart disease is a major contributor to cardiovascular mortality. Endothelin is a potent vasoconstrictive and profibrotic mediator produced by the endothelin-converting enzyme (ECE), whereas natriuretic peptides, degraded by the neutral endopeptidase (NEP), have diuretic, vasodilatory, and antifibrotic properties. Thus, combined ECE/NEP inhibition may halt hypertensive cardiac remodeling. This study examined effects of SLV338, a novel ECE/NEP inhibitor, on cardiac protection in experimental renovascular hypertension (2-kidney, 1-clip [2K1C]). Male rats were allocated to 5 groups: sham-operated rats, untreated animals with 2K1C, 2K1C animals treated with oral SLV338 (30 and 100 mg/kg per day), and 2K1C animals treated with oral losartan (20 mg/kg per day). Treatment duration was 12 weeks. Blood pressure was assessed every 4 weeks. At study end, hearts were taken for histology/computer-aided histomorphometry/immunohistochemistry. Pharmacological properties of SLV338 are described. SLV338 is a dual ECE/NEP inhibitor, as demonstrated both in vitro and in vivo. In the 2K1C study, losartan lowered blood pressure by ≤46 mm Hg, whereas both dosages of SLV338 had no effect. However, SLV338 (both dosages) completely normalized cardiac interstitial fibrosis, perivascular fibrosis, myocyte diameter, and media:lumen ratio of cardiac arteries, as did losartan. Cardiac transforming growth factor-ß1 expression was significantly enhanced in untreated 2K1C rats versus controls, whereas treatment with SLV338 and losartan prevented this effect. Taken together, dual ECE/NEP inhibitor SLV338 prevents cardiac remodeling to the same extent as losartan, but in a blood pressure-independent manner, in a rat model of renovascular hypertension. This effect is at least partially mediated via suppression of cardiac transforming growth factor-ß1 expression.

Nitric oxide-independent stimulation of soluble guanylate cyclase reduces organ damage in experimental low-renin and high-renin models.

OBJECTIVES: The nitric oxide-soluble guanylate cyclase (sGC)-cGMP signal transduction pathway is impaired in different cardiovascular diseases, including pulmonary hypertension, heart failure and arterial hypertension. Riociguat is a novel stimulator of soluble guanylate cyclase (sGC). However, little is known about the effects of sGC stimulators in experimental models of hypertension. We thus investigated the cardio-renal protective effects of riociguat in low-renin and high-renin rat models of hypertension. METHODS: The vasorelaxant effect of riociguat was tested in vitro on isolated saphenous artery rings of normal and nitrate tolerant rabbits. The cardiovascular in-vivo effects of sGC stimulation were evaluated in hypertensive renin-transgenic rats treated with the nitric oxide-synthase inhibitor N-nitro-L-arginine methyl ester (L-NAME) (high-renin model) and in rats with 5/6 nephrectomy (low-renin model). RESULTS: In both animal models, riociguat treatment improved survival and normalized blood pressure. Moreover, in the L-NAME study part, riociguat reduced cardiac target organ damage as indicated by lower plasma ANP, lower relative left ventricular weight and lower cardiac interstitial fibrosis, and reduced renal target organ damage as indicated by lower plasma creatinine and urea, less glomerulosclerosis and less renal interstitial fibrosis. In the 5/6 nephrectomy study part, riociguat reduced cardiac target organ damage as indicated by lower plasma ANP, lower relative left ventricular weight, lower myocyte diameter and lower arterial media/lumen ratio, and reduced renal target organ damage as indicated by improved creatinine clearance and less renal interstitial fibrosis. CONCLUSION: We demonstrate for the first time that the novel sGC stimulator riociguat shows in two independent models of hypertension a potent protection against cardiac and renal target organ damage.

Pulmonary fibrosis in L-NAME-treated mice is dependent on an activated endothelin system.

Activation of the endothelin (ET) system promotes vasoconstriction, inflammation, and fibrosis in various tissues, including the lung. Therefore, ET-1 transgenic mice overexpressing ET-1 develop pulmonary fibrosis in a slow, age-dependent manner. In vivo, NO is the most important counterregulatory mediator of the ET system and decreases ET-1 promoter activity. The aim of our study was to elucidate the impact on pulmonary inflammation and fibrosis of the interaction between NO and the ET system in young ET-1 transgenic mice before the onset of pulmonary fibrosis. Male ET-1 transgenic mice and wild-type littermates at the age of 8 weeks were randomly allocated to the following 6 groups: WT (n = 11), wild-type animals without treatment; WT + L-NAME (n = 14), wild-type animals receiving L-NAME, an inhibitor of NO synthase; WT + L-NAME + LU (n = 13), wild-type animals receiving L-NAME and LU 302872, a dual ETA/ETB-receptor antagonist; ET1tg (n = 10), ET-1 transgenic mice; ET1tg + L-NAME (n = 13); and ET1tg + L-NAME + LU (n = 13). After 6 weeks, animals were euthanized, and hearts and lungs were harvested for histology and immunohistochemistry. No differences in pulmonary inflammation, as indicated by macrophage infiltration, or in interstitial fibrosis were observed between WT and ET1tg mice at baseline; however, inflammation and interstitial fibrosis were significantly enhanced in ET1tg mice, but not in WT groups, after L-NAME treatment. The combined ETA/ETB-receptor antagonist LU 302872 abolished inflammation and interstitial fibrosis in L-NAME-treated ET1tg mice. Perivascular fibrosis and media/lumen ratio of pulmonary bronchi and arteries did not differ between all study groups. In our study L-NAME induced pulmonary fibrosis and inflammation only in young ET1tg mice. Additional treatment with LU 302872 abolished these effects. We thus conclude that an imbalance between an activated ET system and a suppressed NO system contributes to pulmonary inflammation and fibrosis.