Association of Apolipoprotein E4-related Microvascular Disease in the Alzheimer's Disease Hippocampal CA1 Stratum Radiatum.

Current data suggest a hypothesis of vascular pathogenesis for the development and progression of Alzheimer's disease (AD). To investigate this, we studied the association of apolipoprotein E4 (APOE4) gene on microvessels in human autopsy-confirmed AD with and without APOE4, compared with age/sex-matched control (AC) hippocampal CA1 stratum radiatum. AD arterioles (without APOE4 gene) had mild oxidative stress and loss of vascular endothelial growth factor (VEGF) and endothelial cell density, reflecting aging progression. In AD + APOE4, an increase in strong oxidative DNA damage marker 8-hydroxy-2'-deoxyguanosine (8-OHdG), VEGF, and endothelial cell density were associated with increased diameter of arterioles and perivascular space dilation. In cultured human brain microvascular cells (HBMECs), treatment of ApoE4 protein plus amyloid-ß (Aß) oligomers increased superoxide production and the apoptotic marker cleaved caspase 3, sustained hypoxia inducible factor-1α (HIF-1α) stability that was associated with an increase in MnSOD, VEGF, and cell density. This cell over-proliferation was inhibited with the antioxidants N-acetyl cysteine and MnTMPyP, the HIF-1α inhibitor echinomycin, the VEGFR-2 receptor blocker SU1498, the protein kinase C (PKC) ε knock-down (KD) and the extracellular signal-regulated kinase 1/2 (ERK) inhibitor FR180204. The PKCε KD and echinomycin decreased VEGF and/or ERK. In conclusion, AD capillaries and arterioles in hippocampal CA1 stratum radiatum of non-APOE4 carriers are related with aging, while those in APOE4 carriers with AD are related with pathogenesis of cerebrovascular disease.

PKCε activator protects hippocampal microvascular disruption and memory defect in 3×Tg-Alzheimer's disease mice with cerebral microinfarcts.

Background: Current evidence suggests that microvessel disease is involved in Alzheimer's disease (AD). Cerebrovascular disease correlates with cardiovascular disease and is complicated in ≈40% of AD patients. The protein kinase C (PKC) ε activator DCPLA can stimulate human antigen (Hu) R that prevents degradation and promotes the translation of mitochondrial Mn-superoxide dismutase (MnSOD) and vascular endothelial growth factor-A (VEGF) mRNAs. Methods: To induce brain microinfarcts, we injected triple transgenic (3×Tg) and wild-type (WT) control mice with microbeads (20 µm caliber) into common carotid arteries, with or without the DCPLA-ME (methyl-ester) for 2 weeks. After water maze training, mice at 16 months old were examined for confocal immunohistochemistry at a single cell or microvessel level in the hippocampal CA1 area, important for spatial memory storage, and in the dorsal hippocampus by western blots. Results: In 3×Tg mice without cerebral microinfarcts, an accelerating age-related increase in (mild) oxidative stress and hypoxia inducible factor (HIF)-1α, but a reduction in VEGF, mitochondrial transcription factor A (TFAM), and MnSOD were associated with capillary loss. The change was less pronounced in arterioles. However, in 3×Tg mice with cerebral microinfarcts, increasing arteriolar diameter and their wall cells were related with the strong oxidative DNA damage 8-hydroxy-2'-deoxyguanosine (8-OHdG), apoptosis (cleaved caspase 3), and sustained hypoxia (increased HIF-1α and VEGF/PKCε/extracellular signal regulated kinase or ERK pathway). Microocclusion enhanced the loss of the synaptic marker spinophilin, astrocytic number, and astrocyte-vascular coupling areas and demyelination of axons. DCPLA-ME prevented spatial memory defect; strong oxidative stress-related apoptosis; sustained hypoxia (by reducing HIF-1α and VEGF); and exaggerated cell repair in arteriolar walls, pericapillary space dilation, neuro-glial-vascular disruption, and demyelination. Conclusion: In conclusion, in 3×Tg mice with cerebral microinfarcts, sustained hypoxia (increased HIF-1α and VEGF signals) is dominant with arteriolar wall thickening, and DCPLA has a protective effect on sustained hypoxia.

HIV Promotes Neurocognitive Impairment by Damaging the Hippocampal Microvessels.

Current evidence suggests that mild cerebrovascular changes could induce neurodegeneration and contribute to HIV-associated neurocognitive disease (HAND) in HIV patients. We investigated both the quantitative and qualitative impact of HIV infection on brain microvessels, especially on hippocampal microvessels, which are crucial for optimal O2 supply, and thus for maintaining memory and cognitive abilities. The results obtained using cultured human brain microvascular endothelial cells (HBMEC) were reproduced using a suitable mouse model and autopsied human HIV hippocampus. In HBMEC, we found significantly higher oxidative stress-dependent apoptotic cell loss following 5 h of treatment of GST-Tat (1 µg/ml) compared to GST (1 µg/ml) control. We noticed complete recovery of HBMEC cells after 24 h of GST-Tat treatment, due to temporal degradation or inactivation of GST-Tat. Interestingly, we found a sustained increase in mitochondrial oxidative DNA damage marker 8-OHdG, as well as an increase in hypoxia-inducible factor hypoxia-inducible factor-1α (HIF-1α). In our mouse studies, upon short-term injection of GST-Tat, we found the loss of small microvessels (mostly capillaries) and vascular endothelial growth factor (VEGF), but not large microvessels (arterioles and venules) in the hippocampus. In addition to capillary loss, in the post-mortem HIV-infected human hippocampus, we observed large microvessels with increased wall cells and perivascular tissue degeneration. Together, our data show a crucial role of Tat in inducing HIF-1α-dependent inhibition of mitochondrial transcriptional factor A (TFAM) and dilated perivascular space. Thus, our results further define the underlying molecular mechanism promoting mild cerebrovascular disease, neuropathy, and HAND pathogenesis in HIV patients.

PKCε Activation Restores Loss of PKCε, Manganese Superoxide Dismutase, Vascular Endothelial Growth Factor, and Microvessels in Aged and Alzheimer's Disease Hippocampus.

Vascular endothelial dysfunction and capillary loss are currently considered to be a primary phenotype of normal human aging and Alzheimer's disease (AD). Activation of protein kinase C (PKCε) improves several molecular, cellular, physiological, and behavioral endpoints, yet it is not known whether a loss of PKCε activity occurs in the microvascular endothelium in aged and AD hippocampi, whether this loss contributes to microvascular change, or whether activation of PKCε protects against microvascular damage, an early change that induces age-associated memory defect and AD. We investigated the effect of the PKCε activation on microvascular loss in the hippocampus, important for memory storage. In cultured human brain microvascular endothelial cells, tert-butyl hydroperoxide induced oxidative stress and a decrease in manganese superoxide dismutase (MnSOD) mRNA and protein expression that were blocked by the antioxidant drugs. The PKCε activators bryostatin and DCPLA methyl ester increased PKCε, associated with an increase in MnSOD mRNA and its protein as well as vascular endothelial growth factor (VEGF), which was inhibited by the mRNA-stabilizing HuR inhibitors. In rats (>24 months old) and AD transgenic mice Tg2576 (5 months old), bryostatin or DCP-LA prevented a decrease in vascular PKCε, MnSOD, and VEGF and prevented microvascular loss and age-related memory impairment. An autopsy-confirmed AD hippocampus showed a decrease in PKCε and MnSOD mRNAs and their proteins and VEGF as well as in microvascular density compared to non-AD controls. In conclusion, the PKCε activation can rescue a decrease in PKCε, MnSOD, and VEGF via posttranscription regulation and alleviate oxidative stress, and in doing so, prevent microvascular loss during aging and AD.

Resveratrol Improves Recovery and Survival of Diet-Induced Obese Mice Undergoing Extended Major (80%) Hepatectomy.

INTRODUCTION: Loss of hepatic epidermal growth factor receptor (EGFR) expression is a cause for the increased perioperative risk for complications and death in patients with obesity and fatty liver undergoing liver resection. Herein, we set out to identify agents that might increase EGFR expression and improve recovery for patients with fatty liver undergoing resection. Using the diet-induced obese (DIO) mouse model of fatty liver, we examined resveratrol as a therapy to induce EGFR expression and improve outcomes following 80% partial hepatectomy (PH) in a murine model. METHODS: DIO mice were fed resveratrol or carrier control by gavage. EGFR expression and the response to major (80%) PH were examined. RESULTS: Based on an Illumina analysis, resveratrol was identified as increasing EGFR gene expression in A549 cells. Resveratrol was observed to also increase EGFR protein expression in A549 cells. DIO mice fed resveratrol by gavage (75 mg/kg) demonstrated an increased EGFR expression without the identified hepatic toxicity. Resveratrol and control mice subjected to 80% PH, a model of high mortality hepatectomy in DIO mice, demonstrated macroscopically decreased fatty liver and fewer liver hemorrhagic petechiae. Resveratrol pretreatment ameliorated liver injury and accelerated regeneration of the hepatic remnant after 80% PH including decreasing serum ALT and bilirubin, while increasing hepatic PCNA expression. Resveratrol increased induction of p-STAT3 and p-AKT after 80% hepatectomy. Resveratrol pretreatment significantly improved survival rates in DIO mice undergoing extended 80% PH. CONCLUSIONS: Oral resveratrol restores EGFR expression in fatty liver. Resveratrol may be a promising protective agent in instances where extensive hepatic resection of fatty liver is required.

Meloxicam increases epidermal growth factor receptor expression improving survival after hepatic resection in diet-induced obese mice.

BACKGROUND: Patients with fatty liver have delayed regenerative responses, increased hepatocellular injury, and increased risk for perioperative mortality. Currently, no clinical therapy exists to prevent liver failure or improve regeneration in patients with fatty liver. Previously we demonstrated that obese mice have markedly reduced levels of epidermal growth factor receptor in liver. We sought to identify pharmacologic agents to increase epidermal growth factor receptor expression to improve hepatic regeneration in the setting of fatty liver resection. METHODS: Lean (20% calories from fat) and diet-induced obese mice (60% calories from fat) were subjected to 70% or 80% hepatectomy. RESULTS: Using the BaseSpace Correlation Engine of deposited gene arrays we identified agents that increased hepatic epidermal growth factor receptor. Meloxicam was identified as inducing epidermal growth factor receptor expression across species. Meloxicam improved hepatic steatosis in diet-induced obese mice both grossly and histologically. Immunohistochemistry and Western blot analysis demonstrated that meloxicam pretreatment of diet-induced obese mice dramatically increased epidermal growth factor receptor protein expression in hepatocytes. After 70% hepatectomy, meloxicam pretreatment ameliorated liver injury and significantly accelerated mitotic rates of hepatocytes in obese mice. Recovery of liver mass was accelerated in obese mice pretreated with meloxicam (by 26% at 24 hours and 38% at 48 hours, respectively). After 80% hepatectomy, survival was dramatically increased with meloxicam treatment. CONCLUSION: Low epidermal growth factor receptor expression is a common feature of fatty liver disease. Meloxicam restores epidermal growth factor receptor expression in steatotic hepatocytes. Meloxicam pretreatment may be applied to improve outcome after fatty liver resection or transplantation with steatotic graft.

Epidermal growth factor receptor restoration rescues the fatty liver regeneration in mice.

Hepatic steatosis is a common histological finding in obese patients. Even mild steatosis is associated with delayed hepatic regeneration and poor outcomes following liver resection or transplantation. We sought to identify and target molecular pathways that mediate this dysfunction. Lean mice and mice made obese through feeding of a high-fat, hypercaloric diet underwent 70 or 80% hepatectomy. After 70% resection, obese mice demonstrated 100% survival but experienced increased liver injury, reduced energy stores, reduced mitoses, increased necroapoptosis, and delayed recovery of liver mass. Increasing liver resection to 80% was associated with mortality of 40% in lean and 80% in obese mice (P < 0.05). Gene expression profiling showed decreased epidermal growth factor receptor (EGFR) in fatty liver. Meta-analysis of expression studies in mice, rats, and patients also demonstrated reduction of EGFR in fatty liver. In mice, both EGFR and phosphorylated EGFR decreased with increasing percent body fat. Hydrodynamic transfection of EGFR plasmids in mice corrected fatty liver regeneration, reducing liver injury, increasing proliferation, and improving survival after 80% resection. Loss of EGFR expression is rate limiting for liver regeneration in obesity. Therapies directed at increasing EGFR in steatosis might promote liver regeneration and survival following hepatic resection or transplantation.

JAK/STAT3 pathway inhibition blocks skeletal muscle wasting downstream of IL-6 and in experimental cancer cachexia.

Cachexia, the metabolic dysregulation leading to sustained loss of muscle and adipose tissue, is a devastating complication of cancer and other chronic diseases. Interleukin-6 and related cytokines are associated with muscle wasting in clinical and experimental cachexia, although the mechanisms by which they might induce muscle wasting are unknown. One pathway activated strongly by IL-6 family ligands is the JAK/STAT3 pathway, the function of which has not been evaluated in regulation of skeletal muscle mass. Recently, we showed that skeletal muscle STAT3 phosphorylation, nuclear localization, and target gene expression are activated in C26 cancer cachexia, a model with high IL-6 family ligands. Here, we report that STAT3 activation is a common feature of muscle wasting, activated in muscle by IL-6 in vivo and in vitro and by different types of cancer and sterile sepsis. Moreover, STAT3 activation proved both necessary and sufficient for muscle wasting. In C(2)C(12) myotubes and in mouse muscle, mutant constitutively activated STAT3-induced muscle fiber atrophy and exacerbated wasting in cachexia. Conversely, inhibiting STAT3 pharmacologically with JAK or STAT3 inhibitors or genetically with dominant negative STAT3 and short hairpin STAT3 reduced muscle atrophy downstream of IL-6 or cancer. These results indicate that STAT3 is a primary mediator of muscle wasting in cancer cachexia and other conditions of high IL-6 family signaling. Thus STAT3 could represent a novel therapeutic target for the preservation of skeletal muscle in cachexia.

Interleukin-6 is an important in vivo inhibitor of intestinal epithelial cell death in mice.

BACKGROUND AND AIMS: Interleukin-6 (IL-6) is a well-recognised mediator of liver disease and regeneration. However, the in vivo effects of IL-6 on enterocytes and the intestinal tract have not been elucidated. We sought to determine the in vivo effects of IL-6 on enterocytes. METHODS: Murine models of increased or absent IL-6 were examined. RESULTS: Systemic, high-dose IL-6 administration to mice over 7-10 days resulted in intestinal hyperplasia with a approximately 40% increase in small bowel mass and in intestinal villus height. No increase in crypt cell proliferation was noted. IL-6 administration was associated with induction of pSTAT3 in enterocytes along the lower and middle regions of villi but not in crypts. IL-6 administration was also associated with induction of anti-apoptotic proteins including pAKT, and FLIP along with decreased executor caspase activity and PARP cleavage. Pulse bromodeoxyuridine labelling demonstrated equivalent crypt cell proliferation rates but prolonged enterocyte lifespan and slowed enterocyte migration rates in IL-6 treated mice. Furthermore, IL-6 treated mice showed less intestinal injury and improved barrier function following ischaemia reperfusion of the small bowel. Conversely, Il6 null mice exhibited impaired recovery following massive enterectomy and increased apoptosis after 5-fluorouracil chemotherapy relative to wild-type controls. CONCLUSIONS: IL-6 inhibited both constitutive and induced enterocyte cell death in vivo. Loss of IL-6 in mice resulted in increased activation of pro-apoptotic and necrotic pathways in enterocytes after injury. Therapies that augment IL-6 or its signalling pathways may help manage intestinal disorders associated with increased apoptosis, necrosis and gut injury.

Interleukin-6 inhibits oxidative injury and necrosis after extreme liver resection.

UNLABELLED: Extreme hepatectomy or resection of more than 80% of liver mass often leads to liver failure and death and is a major limitation to therapeutic liver resection for patients with liver tumors. We sought to define the mechanisms leading to liver failure and to determine the utility of interleukin-6 (IL-6) administration to improve outcomes. Mice were injected with Chinese hamster ovary cells expressing human IL-6 or no recombinant protein, or were administered recombinant IL-6 or carrier by osmotic mini-pump. Mice were then subjected to 70% or 87% hepatectomy. Light and electron microscopy of liver sections after 87% hepatectomy showed ballooning hepatocytes, vacuolar changes, and mitochondrial abruption, with absence of anoikic nuclei. No significant activation of executor caspases or DNA laddering was observed, although a dramatic decrease in cellular adenosine triphosphate (ATP) stores was measured, suggesting cell death was by a necrotic pathway involving mitochondrial dysfunction. A large increase in protein oxidation was observed, indicative of significant oxidative stress. IL-6 treatment before 87% hepatectomy resulted in less biochemical and histological evidence of liver injury as well as earlier proliferating chain nuclear antigen (PCNA) expression and accelerated recovery of liver mass. IL-6 pretreatment induced the antioxidative injury proteins, ref-1 and GPX1, decreased protein oxidation, vacuolar changes and leakage of mitochondrial products, improved ATP stores, and maintained cellular ultrastructure after 87% hepatectomy. CONCLUSION: Massive oxidative injury and mitochondrial dysfunction occurs in the liver after extreme hepatectomy. IL-6 improves recovery and survival from extreme liver resection by enhancing pro-growth pathways, reducing oxidative stress, and maintaining mitochondrial function.

Paradoxical effects of short- and long-term interleukin-6 exposure on liver injury and repair.

Interleukin-6 (IL-6) is an important mediator of liver regeneration and repair that is also elevated in chronic liver diseases, including fatty liver of obesity and cirrhosis. IL-6 has been reported both to delay and accelerate liver regeneration. We examined the effects on liver injury and regeneration of a continuous administration of exogenous IL-6 to mice by injection of an IL-6-expressing CHO-cell line in athymic nude mice and by osmotic mini-pump delivery of recombinant murine IL-6. Short-term IL-6 administration (1-2 days) accelerated early recovery of liver mass, whereas more long-term administration (5-7 days) markedly impaired liver regeneration. Similarly, short-term IL-6 treatment increased hepatic resistance to the lethal effects of the Fas agonist Jo-2, but on more prolonged IL-6 exposure the Jo-2 resistance vanished. IL-6 administration initially induced expression of the anti-apoptotic proteins Bcl-2 and Bcl-xL, correlating with protection against Fas-mediated cell death. More prolonged IL-6 administration, however, resulted in marked induction of the pro-apoptotic protein Bax. This result coincided with increased activation of the type II or intrinsic, mitochondrial path to cell death, manifested by increased caspase-9 activation and increased cytochrome c release after Jo-2 exposure. These data demonstrate that IL-6 can function acutely to improve hepatic regeneration and repair, but that more chronic exposure not only abolishes the protective effects of IL-6, but actually sensitizes the liver to injury and death. In conclusion, elevated IL-6 in certain chronic liver diseases contributes to an increased likelihood of liver failure after injury.