Endothelial dysfunction and altered mechanical and structural properties of resistance arteries in a murine model of graft-versus-host disease.

A putative involvement of the vasculature seems to play a critical role in the pathophysiology of graft-versus-host disease (GVHD). We aimed to characterize alterations of mesenteric resistance arteries in GVHD in a fully MHC-mismatched model of BALB/c mice conditioned with total body irradiation that underwent transplantation with bone marrow cells and splenocytes from syngeneic (BALB/c) or allogeneic (C57BL/6) donors. After 4 weeks, animals were sacrificed and mesenteric resistance arteries were studied in a pressurized myograph. The expression of endothelial (eNOS) and inducible nitric oxide (NO)-synthase (iNOS) was quantified and vessel wall ultrastructure was investigated with electron microscopy. The myograph study revealed an endothelial dysfunction in allogeneic-transplant recipients, whereas endothelium-independent vasodilation was similar to syngeneic-transplant recipients or untreated controls. The expression of eNOS was decreased and iNOS increased, possibly contributing to endothelial dysfunction. Additionally, arteries of allogeneic transplant recipients exhibited a geometry-independent increase in vessels strain. For both findings, electron microscopy provided a structural correlate by showing severe damage of the whole vessel wall in allogeneic-transplant recipient animals. Our study provides further data to prove, and is the first to characterize, functional and structural vascular alterations in the early course after allogeneic transplantation directly in an ex vivo setting and, therefore, strongly supports the hypothesis of a vascular form of GVHD.

Type 2 diabetes: increased expression and contribution of IKCa channels to vasodilation in small mesenteric arteries of ZDF rats.

Impaired endothelial function, which is dysregulated in diabetes, also precedes hypertension. We hypothesized that in Type 2 diabetes, the impaired endothelium-dependent relaxation is due to a loss of endothelium-derived hyperpolarization (EDH) that is regulated by impaired ion channel function. Zucker diabetic fatty (ZDF), Zucker heterozygote, and homozygote lean control rats were used as the experimental models in our study. Third-order mesenteric arteries were dissected and mounted on a pressure myograph; mRNA was quantified by RT-PCR and channel proteins by Western blotting. Under nitric oxide (NO) synthase and cyclooxygenase inhibition, endothelial stimulation with ACh fully relaxes control but not diabetic arteries. In contrast, when small-conductance calcium-activated potassium (KCa) channels and intermediate- and large-conductance KCa (I/BKCa) are inhibited with apamin and charybdotoxin, NO is able to compensate for ACh-induced relaxation in control but not in diabetic vessels. After replacement of charybdotoxin with 1-[(2-chlorophenyl)diphenylmethyl]-(1)H-pyrazole (TRAM-34; IKCa inhibitor), ACh-induced relaxation in diabetic animals is attenuated. Specific inhibition with TRAM-34 or charybdotoxin attenuates ACh relaxation in diabetes. Stimulation with 1-ethyl-2-benzimidazolinone (IKCa activator) shows a reduced relaxation in diabetes. Activation of BKCa with 1,3-dihydro-1-[2-hydroxy-5-(trifluoromethyl)phenyl]-5-(trifluoromethyl)-(2)H-benzimidazol-2-one NS619 leads to similar relaxations of control and diabetic arteries. RT-PCR and Western blot analysis demonstrate elevated mRNA and protein expression levels of IKCa in diabetes. Our results suggest that the compensatory effect of NO and EDH-associated, endothelium-dependent relaxation is reduced in ZDF rats. Specific blockade of IKCa with TRAM-34 reduces NO and EDH-type relaxation in diabetic rats, indicating an elevated contribution of IKCa in diabetic small mesenteric artery relaxation. This finding correlates with increased IKCa mRNA and protein expression in this vessel.

Experimental heart failure induces alterations of the lung proteome--insight into molecular mechanisms.

BACKGROUND: Heart failure (CHF) is characterized by dyspnea and pulmonary changes. The underlying molecular adaptations are unclear, but might provide targets for therapeutic interventions. We therefore conceived a study to determine molecular changes of early pulmonary stress failure in a model of tachycardia-induced heart failure. METHODS: CHF was induced in rabbits by progessive right ventricular pacing (n=6). Invasive blood pressure measurements and echocardiography were repeatedly performed. Untreated animals served as controls (n=6). Pulmonary tissue specimens were subjected to two-dimensional gel electrophoresis, and differentially expressed proteins were identified by mass spectrometry. Selected proteins were validated by Western Blot analysis and localized by immunohistochemical staining. RESULTS: CHF animals were characterized by significantly altered functional, morphological, and hemodynamic parameters. Upon proteomic profiling, a total of 33 proteins was found to be differentially expressed in pulmonary tissue of CHF animals (18 up-regulated, and 15 down-regulated) belonging to 4 functional groups: 1. proteins involved in maintaining cytoarchitectural integrity, 2. plasma proteins indicating impaired alveolar-capillary permeability, 3. proteins with antioxidative properties, and 4. proteins participating in the metabolism of selenium compounds CONCLUSION: Experimental heart failure profoundly alters the pulmonary proteome. Our results supplement the current knowledge of pulmonary stress failure by specifying its molecular fundament.

Antidiabetic treatment restores adiponectin serum levels and APPL1 expression, but does not improve adiponectin-induced vasodilation and endothelial dysfunction in Zucker diabetic fatty rats.

BACKGROUND: Adiponectin is able to induce NO-dependent vasodilation in Zucker lean (ZL) rats, but this effect is clearly alleviated in their diabetic littermates, the Zucker diabetic fatty (ZDF) rats. ZDF rats also exhibit hypoadiponectinemia and a suppressed expression of APPL1, an adaptor protein of the adiponectin receptors, in mesenteric resistance arteries. Whether an antidiabetic treatment can restore the vasodilatory effect of adiponectin and improve endothelial function in diabetes mellitus type 2 is not known. METHODS: During our animal experiment from week 11 to 22 in each case seven ZDF rats received an antidiabetic treatment with either insulin (ZDF+I) or metformin (ZDF+M). Six normoglycemic ZL and six untreated ZDF rats served as controls. Blood glucose was measured at least weekly and serum adiponectin levels were quantified via ELISA in week 11 and 22. The direct vasodilatory response of their isolated mesenteric resistance arteries to adiponectin as well as the endothelium-dependent and -independent function was evaluated in a small vessel myograph. Additionally, the expression of different components of the adiponectin signaling pathway in the resistance arteries was quantified by real-time RT-PCR. RESULTS: In ZDF rats a sufficient blood glucose control could only be reached by treatment with insulin, but both treatments restored the serum levels of adiponectin and the expression of APPL1 in small resistance arteries. Nevertheless, both therapies were not able to improve the vasodilatory response to adiponectin as well as endothelial function in ZDF rats. Concurrently, a downregulation of the adiponectin receptors 1 and 2 as well as endothelial NO-synthase expression was detected in insulin-treated ZDF rats. Metformin-treated ZDF rats showed a reduced expression of adiponectin receptor 2. CONCLUSIONS: An antidiabetic treatment with either insulin or metformin in ZDF rats inhibits the development of hypoadiponectinemia and downregulation of APPL1 in mesenteric resistance arteries, but is not able to improve adiponectin induced vasodilation and endothelial dysfunction. This is possibly due to alterations in the expression of adiponectin receptors and eNOS.

Expression of fourteen novel obesity-related genes in Zucker diabetic fatty rats.

BACKGROUND: Genome-wide association studies (GWAS) are useful to reveal an association between single nucleotide polymorphisms and different measures of obesity. A multitude of new loci has recently been reported, but the exact function of most of the according genes is not known. The aim of our study was to start elucidating the function of some of these genes. METHODS: We performed an expression analysis of fourteen genes, namely BDNF, ETV5, FAIM2, FTO, GNPDA2, KCTD15, LYPLAL1, MCR4, MTCH2, NEGR1, NRXN3, TMEM18, SEC16B and TFAP2B, via real-time RT-PCR in adipose tissue of the kidney capsule, the mesenterium and subcutaneum as well as the hypothalamus of obese Zucker diabetic fatty (ZDF) and Zucker lean (ZL) rats at an age of 22 weeks. RESULTS: All of our target genes except for SEC16B showed the highest expression in the hypothalamus. This suggests a critical role of these obesity-related genes in the central regulation of energy balance. Interestingly, the expression pattern in the hypothalamus showed no differences between obese ZDF and lean ZL rats. However, LYPLAL1, TFAP2B, SEC16B and FAIM2 were significantly lower expressed in the kidney fat of ZDF than ZL rats. NEGR1 was even lower expressed in subcutaneous and mesenterial fat, while MTCH2 was higher expressed in the subcutaneous and mesenterial fat of ZDF rats. CONCLUSION: The expression pattern of the investigated obesity genes implies for most of them a role in the central regulation of energy balance, but for some also a role in the adipose tissue itself. For the development of the ZDF phenotype peripheral rather than central mechanisms of the investigated genes seem to be relevant.

Acute Renal Graft-Versus-Host Disease in a Murine Model of Allogeneic Bone Marrow Transplantation.

Acute kidney injury (AKI) is a very common complication after allogeneic bone marrow transplantation (BMT) and is associated with a poor prognosis. Generally, the kidneys are assumed to not be no direct targets of graft-versus-host disease (GvHD), and renal impairment is often attributed to several other factors occurring in the early phase after BMT. Our study aimed to prove the existence of renal GvHD in a fully major histocompatibility complex (MHC)-mismatched model of BALB/c mice conditioned and transplanted according to 2 different intensity protocols. Syngeneically transplanted and untreated animals served as controls. Four weeks after transplantation, allogeneic animals developed acute GvHD that was more pronounced in the high-intensity protocol (HIP) group than in the low-intensity protocol (LIP) group. Urea and creatinine as classic serum markers of renal function could not verify renal impairment 4 weeks after BMT. Creatinine levels were even reduced as a result of catabolic metabolism and loss of muscle mass due to acute GvHD. Proteinuria, albuminuria, and urinary N-acetyl-beta-d-glucosaminidase (NAG) levels were measured as additional renal markers before and after transplantation. Albuminuria and NAG were only significantly increased after allogeneic transplantation, correlating with disease severity between HIP and LIP animals. Histological investigations of the kidneys showed renal infiltration of T cells and macrophages with endarteriitis, interstitial nephritis, tubulitis, and glomerulitis. T cells consisted of CD4+, CD8+, and FoxP3+ cells. Renal expression analysis of allogeneic animals showed increases in indoleamine-2,3 dioxygenase (IDO), different cytokines (tumor necrosis factor α, interferon-γ, interleukin 1 α [IL-1α], IL-2, IL-6, and IL-10), and adhesion molecules (intercellular adhesion molecule 1 and vascular cell adhesion molecule 1), resembling findings from other tissues in acute GvHD. In summary, our study supports the entity of renal GvHD with histological features suggestive of cell-mediated renal injury. Albuminuria and urinary NAG levels may serve as early markers of renal impairment.

Combined Inhibition of the Renin-Angiotensin System and Neprilysin Positively Influences Complex Mitochondrial Adaptations in Progressive Experimental Heart Failure.

BACKGROUND: Inhibitors of the renin angiotensin system and neprilysin (RAS-/NEP-inhibitors) proved to be extraordinarily beneficial in systolic heart failure. Furthermore, compelling evidence exists that impaired mitochondrial pathways are causatively involved in progressive left ventricular (LV) dysfunction. Consequently, we aimed to assess whether RAS-/NEP-inhibition can attenuate mitochondrial adaptations in experimental heart failure (HF). METHODS AND RESULTS: By progressive right ventricular pacing, distinct HF stages were induced in 15 rabbits, and 6 animals served as controls (CTRL). Six animals with manifest HF (CHF) were treated with the RAS-/NEP-inhibitor omapatrilat. Echocardiographic studies and invasive blood pressure measurements were undertaken during HF progression. Mitochondria were isolated from LV tissue, respectively, and further worked up for proteomic analysis using the SWATH technique. Enzymatic activities of citrate synthase and the electron transfer chain (ETC) complexes I, II, and IV were assessed. Ultrastructural analyses were performed by transmission electron microscopy. During progression to overt HF, intricate expression changes were mainly detected for proteins belonging to the tricarboxylic acid cycle, glucose and fat metabolism, and the ETC complexes, even though ETC complex I, II, or IV enzymatic activities were not significantly influenced. Treatment with a RAS-/NEP-inhibitor then reversed some maladaptive metabolic adaptations, positively influenced the decline of citrate synthase activity, and altered the composition of each respiratory chain complex, even though this was again not accompanied by altered ETC complex enzymatic activities. Finally, ultrastructural evidence pointed to a reduction of autophagolytic and degenerative processes with omapatrilat-treatment. CONCLUSIONS: This study describes complex adaptations of the mitochondrial proteome in experimental tachycardia-induced heart failure and shows that a combined RAS-/NEP-inhibition can beneficially influence mitochondrial key pathways.

Vascular Alterations in a Murine Model of Acute Graft-Versus-Host Disease Are Associated with Decreased Serum Levels of Adiponectin and an Increased Activity and Vascular Expression of Indoleamine 2,3-Dioxygenase.

Graft-versus-host disease (GVHD) is the limiting complication after bone marrow transplantation (BMT), and its pathophysiology seems to be highly influenced by vascular factors. Our study aimed at elucidating possible mechanisms involved in vascular GVHD. For this purpose, we used a fully MHC-mismatched model of BALB/c mice conditioned according to two different intensity protocols with total body irradiation and transplantation of allogeneic (C57BL/6) or syngeneic bone marrow cells and splenocytes. Mesenteric resistance arteries were studied in a pressurized myograph. We also quantified the expression of indoleamine 2,3-dioxygenase (IDO), endothelial (eNOS), and inducible NO synthase (iNOS), as well as several pro- and anti-inflammatory cytokines. We measured the serum levels of tryptophan (trp) and kynurenine (kyn), the kyn/trp ratio (KTR) as a marker of IDO activity, and adiponectin (APN). The myographic study showed a correlation of GVHD severity after allogeneic BMT with functional vessel alterations that started with increased vessel stress and ended in eccentric vessel remodeling, increased vessel strain, and endothelial dysfunction. These alterations were accompanied by increasing IDO activity and decreasing APN levels in the serum of allogeneic animals. The mRNA expression showed significantly elevated IDO, decreased eNOS, and elevation of most studied pro- and anti-inflammatory cytokines. Our study provides further data supporting the importance of vessel alterations in GVHD and is the first to show an association of vascular GVHD with hypoadiponectinemia and an increased activity and vascular expression of IDO. Whether there is also a causative involvement of these two factors in the development of GVHD needs to be further investigated.

Free fatty acids, lipopolysaccharide and IL-1α induce adipocyte manganese superoxide dismutase which is increased in visceral adipose tissues of obese rodents.

Excess fat storage in adipocytes is associated with increased generation of reactive oxygen species (ROS) and impaired activity of antioxidant mechanisms. Manganese superoxide dismutase (MnSOD) is a mitochondrial enzyme involved in detoxification of ROS, and objective of the current study is to analyze expression and regulation of MnSOD in obesity. MnSOD is increased in visceral but not subcutaneous fat depots of rodents kept on high fat diets (HFD) and ob/ob mice. MnSOD is elevated in visceral adipocytes of fat fed mice and exposure of differentiating 3T3-L1 cells to lipopolysaccharide, IL-1α, saturated, monounsaturated and polyunsaturated free fatty acids (FFA) upregulates its level. FFA do not alter cytochrome oxidase 4 arguing against overall induction of mitochondrial enzymes. Upregulation of MnSOD in fat loaded cells is not mediated by IL-6, TNF or sterol regulatory element binding protein 2 which are induced in these cells. MnSOD is similarly abundant in perirenal fat of Zucker diabetic rats and non-diabetic animals with similar body weight and glucose has no effect on MnSOD in 3T3-L1 cells. To evaluate whether MnSOD affects adipocyte fat storage, MnSOD was knocked-down in adipocytes for the last three days of differentiation and in mature adipocytes. Knock-down of MnSOD does neither alter lipid storage nor viability of these cells. Heme oxygenase-1 which is induced upon oxidative stress is not altered while antioxidative capacity of the cells is modestly reduced. Current data show that inflammation and excess triglyceride storage raise adipocyte MnSOD which is induced in epididymal adipocytes in obesity.

Globular and full-length adiponectin induce NO-dependent vasodilation in resistance arteries of Zucker lean but not Zucker diabetic fatty rats.

BACKGROUND: Adiponectin increases nitric oxide (NO) production in endothelial cell cultures and is reduced in the circulation of obese and diabetic patients, but its functional effect on resistance arteries is not yet studied in detail. METHODS: We assessed the direct vasodilatory response of isolated mesenteric resistance arteries of Zucker diabetic fatty (ZDF) rats and Zucker lean (ZL) rats to globular adiponectin (gAd) and full-length adiponectin (fAd) and tested the effect of additional reactive oxygen species (ROS) inhibitors in vitro. Serum adiponectin and insulin levels were measured by ELISA. The mRNA expressions of the adiponectin receptors and the downstream signaling molecules adaptor protein, phosphotyrosine interaction, PH domain and leucine zipper containing 1 (APPL1), adaptor protein, phosphotyrosine interaction, PH domain and leucine zipper containing 2 (APPL2), and endothelial NO synthase (eNOS) in mesenteric resistance arteries were quantified by real-time reverse transcriptase PCR. RESULTS: Both gAd and fAd induced a relevant dose-dependent vasodilation in ZL, but not in hypoadiponectinemic ZDF rats. This effect was totally blunted by L-nitroarginine-methyl-ester indicating NO dependency. The addition of ROS inhibitors could not improve the vasodilatory effect of adiponectin. Vasodilatory response to acetylcholine was reduced in ZDF rats, which could not be enhanced by low-dose adiponectin. Adiponectin receptor 1 (AdipoR1) was higher expressed than adiponectin receptor 2 (AdipoR2) with no significant differences between both animal groups, but APPL1 was significantly decreased in ZDF rats. The eNOS expression was not significantly different between ZL and ZDF rats. CONCLUSIONS: Adiponectin exerts a NO-dependent vasodilation in resistance arteries of normoglycemic ZL rats, but not diabetic ZDF rats. This may contribute to endothelial dysfunction in ZDF rats. Alterations in the expression of APPL1 may be involved in the observed insensitivity to adiponectin in ZDF rats.