Atsushi Miyanohara

Cardiac-specific overexpression of caveolin-3 induces endogenous cardiac protection by mimicking ischemic preconditioning

Background— Caveolae, lipid-rich microdomains of the sarcolemma, localize and enrich cardiac-protective signaling molecules. Caveolin-3 (Cav-3), the dominant isoform in cardiac myocytes, is a determinant of caveolar formation. We hypothesized that cardiac myocyte–specific overexpression of Cav-3 would enhance the formation of caveolae and augment cardiac protection in vivo. Methods and Results— Ischemic preconditioning in vivo increased the formation of caveolae. Adenovirus for Cav-3 increased caveolar formation and phosphorylation of survival kinases in cardiac myocytes. A transgenic mouse with cardiac myocyte–specific overexpression of Cav-3 (Cav-3 OE) showed enhanced formation of caveolae on the sarcolemma. Cav-3 OE mice subjected to ischemia/reperfusion injury had a significantly reduced infarct size relative to transgene-negative mice. Endogenous cardiac protection in Cav-3 OE mice was similar to wild-type mice undergoing ischemic preconditioning; no increased protection was observed in preconditioned Cav-3 OE mice. Cav-3 knockout mice did not show endogenous protection and showed no protection in response to ischemic preconditioning. Cav-3 OE mouse hearts had increased basal Akt and glycogen synthase kinase-3β phosphorylation comparable to wild-type mice exposed to ischemic preconditioning. Wortmannin, a phosphoinositide 3-kinase inhibitor, attenuated basal phosphorylation of Akt and glycogen synthase kinase-3β and blocked cardiac protection in Cav-3 OE mice. Cav-3 OE mice had improved functional recovery and reduced apoptosis at 24 hours of reperfusion. Conclusions— Expression of caveolin-3 is both necessary and sufficient for cardiac protection, a conclusion that unites long-standing ultrastructural and molecular observations in the ischemic heart. The present results indicate that increased expression of caveolins, apparently via actions that depend on phosphoinositide 3-kinase, has the potential to protect hearts exposed to ischemia/reperfusion injury.

Increased smooth muscle cell expression of caveolin-1 and caveolae contribute to the pathophysiology of idiopathic pulmonary arterial hypertension

Vasoconstriction and vascular medial hypertrophy, resulting from increased intracellular [Ca2+] in pulmonary artery smooth muscle cells (PASMC), contribute to elevated vascular resistance in patients with idiopathic pulmonary arterial hypertension (IPAH). Caveolae, microdomains within the plasma membrane, contain the protein caveolin, which binds certain signaling molecules. We tested the hy‐pothesis that PASMC from IPAH patients express more caveolin‐1 (Cav‐1) and caveolae, which contribute to increased capacitative Ca2+ entry (CCE) and DNA synthesis. Immunohistochemistry showed increased expression of Cav‐1 in smooth muscle cells but not endothelial cells of pulmonary arteries from patients with IPAH. Subcellular fractionation and electron microscopy confirmed the increase in Cav‐1 and caveolae expression in IPAH‐PASMC. Treatment of IPAH‐PASMC with agents that deplete membrane cholesterol (methyl‐β‐cyclodextrin or lovastatin) disrupted caveo‐lae, attenuated CCE, and inhibited DNA synthesis of IPAH‐PASMC. Increasing Cav‐1 expression of normal PASMC with a Cav‐1‐encoding adenovirus increased caveolae formation, CCE, and DNA synthesis;treatment of IPAH‐PASMC with siRNA targeted to Cav‐1 produced the opposite effects. Treatments that down‐regulate caveolin/caveolae expression, including cho‐lesterol‐lowering drugs, reversed the increased CCE and DNA synthesis in IPAH‐PASMC. Increased caveolin and caveolae expression thus contribute to IPAH‐PASMC pathophysiology. The close relationship between caveolin/caveolae expression and altered cell physiology in IPAH contrast with previous results obtained in various animal models, including caveolin‐knockout mice, thus emphasizing unique features of the human disease. The results imply that disruption of caveolae in PASMC may provide a novel therapeutic approach to attenuate disease manifestations of IPAH.—Patel H. H., Zhang, S., Murray, F., Suda, R. Y. S., Head, B. P., Yokoyama, U., Swaney, J. S., Niesman, I. R., Schermuly, R. T., Savai Pullamsetti, S., Thistlethwaite, P. A., Miyanohara, A., Farquhar M. G., Yuan J. X.‐J., Insel P. A. Increased smooth muscle cell expression of caveolin‐1 and caveolae contribute to the pathophysiology of idiopathic pulmonary arterial hypertension. FASEB J. 21, 2970–2979 (2007)

Loss of Caveolin-1 Accelerates Neurodegeneration and Aging

Background The aged brain exhibits a loss in gray matter and a decrease in spines and synaptic densities that may represent a sequela for neurodegenerative diseases such as Alzheimers. Membrane/lipid rafts (MLR), discrete regions of the plasmalemma enriched in cholesterol, glycosphingolipids, and sphingomyelin, are essential for the development and stabilization of synapses. Caveolin-1 (Cav-1), a cholesterol binding protein organizes synaptic signaling components within MLR. It is unknown whether loss of synapses is dependent on an age-related loss of Cav-1 expression and whether this has implications for neurodegenerative diseases such as Alzheimers disease. Methodology/Principal Findings We analyzed brains from young (Yg, 3-6 months), middle age (Md, 12 months), aged (Ag, >18 months), and young Cav-1 KO mice and show that localization of PSD-95, NR2A, NR2B, TrkBR, AMPAR, and Cav-1 to MLR is decreased in aged hippocampi. Young Cav-1 KO mice showed signs of premature neuronal aging and degeneration. Hippocampi synaptosomes from Cav-1 KO mice showed reduced PSD-95, NR2A, NR2B, and Cav-1, an inability to be protected against cerebral ischemia-reperfusion injury compared to young WT mice, increased Aβ, P-Tau, and astrogliosis, decreased cerebrovascular volume compared to young WT mice. As with aged hippocampi, Cav-1 KO brains showed significantly reduced synapses. Neuron-targeted re-expression of Cav-1 in Cav-1 KO neurons in vitro decreased Aβ expression. Conclusions Therefore, Cav-1 represents a novel control point for healthy neuronal aging and loss of Cav-1 represents a non-mutational model for Alzheimers disease.

Human Oxidation-Specific Antibodies Reduce Foam Cell Formation and Atherosclerosis Progression

OBJECTIVES We sought to assess the in vivo importance of scavenger receptor (SR)-mediated uptake of oxidized low-density lipoprotein (OxLDL) in atherogenesis and to test the efficacy of human antibody IK17-Fab or IK17 single-chain Fv fragment (IK17-scFv), which lacks immunologic properties of intact antibodies other than the ability to inhibit uptake of OxLDL by macrophages, to inhibit atherosclerosis. BACKGROUND The unregulated uptake of OxLDL by macrophage SR contributes to foam cell formation, but the importance of this pathway in vivo is uncertain. METHODS Cholesterol-fed low-density lipoprotein receptor knockout (LDLR(-/-)) mice were treated with intraperitoneal infusion of human IK17-Fab (2.5 mg/kg) 3 times per week for 14 weeks. Because anti-human antibodies developed in these mice, LDLR(-/-)/low-density lipoprotein receptor Rag 1 double-knockout mice (lacking the ability to make immunoglobulins due to loss of T- and B-cell function) were treated with an adenoviral vector encoding adenovirus expressed (Adv)-IK17-scFv or control adenovirus-enhanced green fluorescent protein vector intravenously every 2 weeks for 16 weeks. RESULTS In LDLR(-/-) mice, infusion of IK17-Fab was able to sustain IK17 plasma levels for the first 8 weeks, but these diminished afterward due to increasing murine anti-IK17 antibody titers. Despite this, after 14 weeks, a 29% decrease in en face atherosclerosis was noted compared with phosphate-buffered saline-treated mice. In LDLR(-/-)/low-density lipoprotein receptor Rag 1 double-knockout mice, sustained levels of plasma IK17-scFv was achieved by Adv-IK17-scFv-mediated hepatic expression, which led to a 46% reduction (p < 0.001) in en face atherosclerosis compared with adenovirus-enhanced green fluorescent protein vector. Importantly, peritoneal macrophages isolated from Adv-IK17-scFv treated mice had decreased lipid accumulation compared with adenovirus-enhanced green fluorescent protein-treated mice. CONCLUSIONS These data support an important role for SR-mediated uptake of OxLDL in the pathogenesis of atherosclerosis and demonstrate that oxidation-specific antibodies reduce the progression of atherosclerosis, suggesting their potential in treating cardiovascular disease in humans.

A Transgenic Minipig Model of Huntington's Disease

BACKGROUND Some promising treatments for Huntingtons disease (HD) may require pre-clinical testing in large animals. Minipig is a suitable species because of its large gyrencephalic brain and long lifespan. OBJECTIVE To generate HD transgenic (TgHD) minipigs encoding huntingtin (HTT)1-548 under the control of human HTT promoter. METHODS Transgenesis was achieved by lentiviral infection of porcine embryos. PCR assessment of gene transfer, observations of behavior, and postmortem biochemical and immunohistochemical studies were conducted. RESULTS One copy of the human HTT transgene encoding 124 glutamines integrated into chromosome 1 q24-q25 and successful germ line transmission occurred through successive generations (F0, F1, F2 and F3 generations). No developmental or gross motor deficits were noted up to 40 months of age. Mutant HTT mRNA and protein fragment were detected in brain and peripheral tissues. No aggregate formation in brain up to 16 months was seen by AGERA and filter retardation or by immunostaining. DARPP32 labeling in WT and TgHD minipig neostriatum was patchy. Analysis of 16 month old sibling pairs showed reduced intensity of DARPP32 immunoreactivity in neostriatal TgHD neurons compared to those of WT. Compared to WT, TgHD boars by one year had reduced fertility and fewer spermatozoa per ejaculate. In vitro analysis revealed a significant decline in the number of WT minipig oocytes penetrated by TgHD spermatozoa. CONCLUSIONS The findings demonstrate successful establishment of a transgenic model of HD in minipig that should be valuable for testing long term safety of HD therapeutics. The emergence of HD-like phenotypes in the TgHD minipigs will require more study.

Mitochondria-localized caveolin in adaptation to cellular stress and injury

We show here that the apposition of plasma membrane caveolae and mitochondria (first noted in electron micrographs >50 yr ago) and caveolae‐mitochondria interaction regulates adaptation to cellular stress by modulating the structure and function of mitochondria. In C57Bl/6 mice engineered to overexpress caveolin specifically in cardiac myocytes (Cav‐3 OE), localization of caveolin to mitochondria increases membrane rigidity (4.2%; P<0.05), tolerance to calcium, and respiratory function (72% increase in state 3 and 23% increase in complex IV activity; P<0.05), while reducing stress‐induced generation of reactive oxygen species (by 20% in cellular superoxide and 41 and 28% in mitochondrial superoxide under states 4 and 3, respectively; P<0.05) in Cav‐3 OE vs. TGneg. By contrast, mitochondrial function is abnormal in caveolin‐knockout mice and Caenorhabditis elegans with null mutations in caveolin (60% increase free radical in Cav‐2 C. elegans mutants; P<0.05). In human colon cancer cells, mitochondria with increased caveolin have a 30% decrease in apoptotic stress (P<0.05), but cells with disrupted mitochondria‐caveolin interaction have a 30% increase in stress response (P<0.05). Targeted gene transfer of caveolin to mitochondria in C57Bl/6 mice increases cardiac mitochondria tolerance to calcium, enhances respiratory function (increases of 90% state 4, 220% state 3, 88% complex IV activity; P<0.05), and decreases (by 33%) cardiac damage (P<0.05). Physical association and apparently the transfer of caveolin between caveolae and mitochondria is thus a conserved cellular response that confers protection from cellular damage in a variety of tissues and settings.—Fridolfsson, H. N., Kawaraguchi, Y., Ali, S. S., Panneerselvam, M., Niesman, I. R., Finley, J. C., Kellerhals, S. E., Migita, M. Y., Okada, H., Moreno, A. L., Jennings, M., Kidd, M. W., Bonds, J. A., Balijepalli, R. C., Ross, R. S., Patel, P. M., Miyanohara, A., Chen, Q., Lesnefsky, E. J., Head, B. P., Roth, D. M., Insel, P. A., Patel, H. H. Mitochondria‐localized caveolin in adaptation to cellular stress and injury. FASEB J. 26, 4637–4649 (2012). www.fasebj.org

Neuron-targeted Caveolin-1 Protein Enhances Signaling and Promotes Arborization of Primary Neurons

Decreased expression of prosurvival and progrowth-stimulatory pathways, in addition to an environment that inhibits neuronal growth, contribute to the limited regenerative capacity in the central nervous system following injury or neurodegeneration. Membrane/lipid rafts, plasmalemmal microdomains enriched in cholesterol, sphingolipids, and the protein caveolin (Cav) are essential for synaptic development/stabilization and neuronal signaling. Cav-1 concentrates glutamate and neurotrophin receptors and prosurvival kinases and regulates cAMP formation. Here, we show that primary neurons that express a synapsin-driven Cav-1 vector (SynCav1) have increased raft formation, neurotransmitter and neurotrophin receptor expression, NMDA- and BDNF-mediated prosurvival kinase activation, agonist-stimulated cAMP formation, and dendritic growth. Moreover, expression of SynCav1 in Cav-1 KO neurons restores NMDA- and BDNF-mediated signaling and enhances dendritic growth. The enhanced dendritic growth occurred even in the presence of inhibitory cytokines (TNFα, IL-1β) and myelin-associated glycoproteins (MAG, Nogo). Targeting of Cav-1 to neurons thus enhances prosurvival and progrowth signaling and may be a novel means to repair the injured and neurodegenerative brain.

Volatile Anesthetics Protect Cancer Cells against Tumor Necrosis Factor-related Apoptosis-inducing Ligand-induced Apoptosis via Caveolins

Background: Volatile anesthetics have a dual effect on cell survival dependent on caveolin expression. The effect of volatile anesthetics on cancer cell survival and death after anesthetic exposure has not been well investigated. The authors examined the effects of isoflurane exposure on apoptosis and its regulation by caveolin-1 (Cav-1). Methods: The authors exposed human colon cancer cell lines to isoflurane and proapoptotic stimuli and assessed what role Cav-1 plays in cell protection. They evaluated apoptosis using assays for nucleosomal fragmentation, cleaved caspase 3 expression, and caspase activity assays. To test the mechanism, they used pharmacologic inhibitors (i.e., pertussis toxin) and assessed changes in glycolysis. Results: Apoptosis as measured by nucleosomal fragmentation was enhanced by isoflurane (1.2% in air) in HT29 (by 64% relative to control, P < 0.001) and decreased in HCT116 (by 23% relative to control, P < 0.001) cells. Knockdown of Cav-1 in HCT116 cells increased the sensitivity to apoptotic stimuli but not with scrambled small interfering RNA (siRNA) treatment (19.7 ± 0.4 vs. 20.0 ± 0.6, P = 0.7786 and 19.7 ± 0.5 vs. 16.3 ± 0.4, P = 0.0012, isoflurane vs. control in Cav-1 small interfering RNA vs. scrambled small interfering RNA treated cells, respectively). The protective effect of isoflurane with various exposure times on apoptosis was enhanced in HT29 cells overexpressing Cav-1 (P < 0.001 by two-way ANOVA). Pertussis toxin effectively blocked the antiapoptotic effect of isoflurane exhibited by Cav-1 in all cell lines. Cav-1 cells had increased glycolysis with isoflurane exposure; however, in the presence of tumor necrosis factor-related apoptosis-inducing ligand, this increase in glycolysis was maintained in HT29-Cav-1 but not control cells. Conclusion: Brief isoflurane exposure leads to resistance against apoptosis via a Cav-1–dependent mechanism.

Separable Mechanisms of Attachment and Cell Uptake during Retrovirus Infection

ABSTRACT In the absence of viral envelope gene expression, cells expressing human immunodeficiency virus type 1 (HIV-1) gag andpol, accessory HIV functions, and a vector genome RNA produce and secrete large amount of noninfectious virus-like particles (VLPs) into the conditioned medium. After partial purification, such HIV-1 VLPs can be made infectious in cell-free conditions in vitro by complex formation with lipofection reagents or with the G protein of vesicular stomatitis virus (VSV-G). The resulting in vitro-modified HIV-1 particles are able to infect nondividing cells. Infectivity of envelope-free HIV VLPs can also be induced by prior modification of target cells through exposure to partially purified VSV-G vesicles. Similarly, infection can be carried out by attachment of envelope-free noninfectious VLPs to unmodified cells followed by subsequent treatment of cells with VSV-G. We interpret these findings to indicate that interaction between a viral envelope and a cell surface receptor is not necessary for the initial virus binding to the cells but is required for subsequent cell entry and infection.

Adenylyl Cyclase Type VI Increases Akt Activity and Phospholamban Phosphorylation in Cardiac Myocytes

Increased expression of adenylyl cyclase VI has beneficial effects on the heart, but strategies that increase cAMP production in cardiac myocytes usually are harmful. Might adenylyl cyclase VI have beneficial effects unrelated to increased β-adrenergic receptor-mediated signaling? We previously reported that adenylyl cyclase VI reduces cardiac phospholamban expression. Our focus in the current studies is how adenylyl cyclase VI influences phospholamban phosphorylation. In cultured cardiac myocytes, increased expression of adenylyl cyclase VI activates Akt by phosphorylation at serine 473 and threonine 308 and is associated with increased nuclear phospho-Akt. Activated Akt phosphorylates phospholamban, a process that does not require β-adrenergic receptor stimulation or protein kinase A activation. These previously unrecognized signaling events would be predicted to promote calcium handling and increase contractile function of the intact heart independently of β-adrenergic receptor activation. We speculate that phospholamban phosphorylation, through activation of Akt, may be an important mechanism by which adenylyl cyclase VI increases the function of the failing heart.