J Cameron Finley's research while affiliated with University of California, San Diego and other places

Thy-1-negative lung fibroblasts are resistant to apoptosis. The mechanisms governing this process and its relevance to fibrotic remodeling remain poorly understood. By using either sorted or transfected lung fibroblasts, we found that Thy-1 expression is associated with downregulation of anti-apoptotic molecules Bcl-2 and Bcl-xL, as well as increased levels of cleaved caspase-9. Addition of rhFasL and staurosporine, well-known apoptosis inducers, caused significantly increased cleaved caspase-3, -8, and PARP in Thy-1-transfected cells. Furthermore, rhFasL induced Fas translocation into lipid rafts and its colocalization with Thy-1. These in vitro results indicate that Thy-1, in a manner dependent upon its glycophosphatidylinositol anchor and lipid raft localization, regulates apoptosis in lung fibroblasts via Fas-, Bcl-, and caspase-dependent pathways. In vivo, Thy-1 deficient (Thy1−/−) mice displayed persistence of myofibroblasts in the resolution phase of bleomycin-induced fibrosis, associated with accumulation of collagen and failure of lung fibrosis resolution. Apoptosis of myofibroblasts is decreased in Thy1−/− mice in the resolution phase. Collectively, these findings provide new evidence regarding the role and mechanisms of Thy-1 in initiating myofibroblast apoptosis that heralds the termination of the reparative response to bleomycin-induced lung injury. Understanding the mechanisms regulating fibroblast survival/apoptosis should lead to novel therapeutic interventions for lung fibrosis.

Caveolae are a nexus for protective signaling. Trafficking of caveolin to mitochondria is essential for adaptation to cellular stress though the trafficking mechanisms remain unknown. The authors hypothesized that G protein-coupled receptor/inhibitory G protein (Gi) activation leads to caveolin trafficking to mitochondria. Mice were exposed to isoflurane or oxygen vehicle (30 min, ±36 h pertussis toxin pretreatment, an irreversible Gi inhibitor). Caveolin trafficking, cardioprotective "survival kinase" signaling, mitochondrial function, and ultrastructure were assessed. Isoflurane increased cardiac caveolae (n = 8 per group; data presented as mean ± SD for Ctrl versus isoflurane; [caveolin-1: 1.78 ± 0.12 vs. 3.53 ± 0.77; P < 0.05]; [caveolin-3: 1.68 ± 0.29 vs. 2.67 ± 0.46; P < 0.05]) and mitochondrial caveolin levels (n = 16 per group; [caveolin-1: 0.87 ± 0.18 vs. 1.89 ± .19; P < 0.05]; [caveolin-3: 1.10 ± 0.29 vs. 2.26 ± 0.28; P < 0.05]), and caveolin-enriched mitochondria exhibited improved respiratory function (n = 4 per group; [state 3/complex I: 10.67 ± 1.54 vs. 37.6 ± 7.34; P < 0.05]; [state 3/complex II: 37.19 ± 4.61 vs. 71.48 ± 15.28; P < 0.05]). Isoflurane increased phosphorylation of survival kinases (n = 8 per group; [protein kinase B: 0.63 ± 0.20 vs. 1.47 ± 0.18; P < 0.05]; [glycogen synthase kinase 3β: 1.23 ± 0.20 vs. 2.35 ± 0.20; P < 0.05]). The beneficial effects were blocked by pertussis toxin. Gi proteins are involved in trafficking caveolin to mitochondria to enhance stress resistance. Agents that target Gi activation and caveolin trafficking may be viable cardioprotective agents.

Microglia are ramified cells that serve as central nervous system (CNS) guardians, capable of proliferation, migration, and generation of inflammatory cytokines. In non-pathological states, these cells exhibit ramified morphology with processes intermingling with neurons and astrocytes. Under pathological conditions, they acquire a rounded amoeboid morphology and proliferative and migratory capabilities. Such morphological changes require cytoskeleton rearrangements. The molecular control points for polymerization states of microtubules and actin are still under investigation. Caveolins (Cav), membrane/lipid raft proteins, are expressed in inflammatory cells, yet the role of Caveolin isoforms in microglia physiology is debatable. We propose caveolins provide a necessary control point in the regulation of cytoskeletal dynamics, and thus investigated a role for caveolins in microglia biology. We detected mRNA and protein for both Cav-1 and Cav-3. Cav-1 protein was significantly less and localized to plasmalemma (PM) and cytoplasmic vesicles (CV) in the microglial inactive state, while the active (amoeboid-shaped) microglia exhibited increased Cav-1 expression. In contrast, Cav-3 was highly expressed in the inactive state and localized with cellular processes and perinuclear regions and was detected in active amoeboid microglia. Pharmacological manipulation of the cytoskeleton in the active or non-active state altered caveolin expression. Additionally, increased Cav-1 expression also increased mitochondrial respiration, suggesting possible regulatory roles in cell metabolism necessary to facilitate the morphological changes. The present findings strongly suggest that regulation of microglial morphology and activity are in part due to caveolin isoforms, providing promising novel therapeutic targets in CNS injury or disease.

Scope: The flavanol (-)-epicatechin (Epi), a component of cacao, has cardiac protective benefits in humans. Our previous study demonstrated Epi has δ-opioid receptor (DOR) binding activity and promotes cardiac protection. Here we examined the effects of 10 days of Epi treatment on: cardiac mitochondrial respiration, reactive oxygen species production, calcium swelling, and mitochondrial membrane fluidity. Methods and results: Mice were randomized into four groups: (i) control (saline), (ii) naltrindole (Nalt; DOR antagonist), (iii) Epi, and (iv) Epi + Nalt and received 1 mg/kg Epi or water via oral gavage. Nalt groups received 5 mg/kg ip per day for 10 days. Significant increases in mitochondrial respiration and enhanced free radical production during state 3 respiration were observed with Epi. Additionally, we observed significant increases in rigidity of mitochondrial membranes and resistance to calcium-induced mitochondrial swelling with Epi treatment. Blocking the DOR with Nalt resulted in decreases in all of the observed parameters by Epi treatment. Conclusion: These findings indicate that Epi induces an integrated response that includes metabolic and structural changes in cardiac mitochondria resulting in greater functional capacity via DOR. Mitochondrial targeted effects of epicatechin may explain the physiologic benefit observed on cardiac protection and support epicatechin's potential clinical application as a cardiac protective mimetic.

Diabetes is a worldwide epidemic with cardiovascular disease being a major complication. Caveolins act as scaffolding molecules for regulating signaling. Overexpression of caveolin protects the heart from cardiovascular stress. We hypothesize that cardiac‐specific caveolin‐3 (Cav‐3) overexpression (OE) will protect the diabetic heart. Transgene negative (TGneg) or Cav‐3 OE mice were given a single dose of streptozotocin (75mg/kg) and then placed on a high fat diet to induce type II diabetes mellitus (T2DM). After 3 months, TGneg T2DM mice and Cav‐3 OE T2DM mice showed an increase in body weight, altered glucose tolerance response, and increased insulin levels compared to controls on normal diet. Cav‐3 OE T2DM mitochondria showed protection from calcium swelling and reactive oxygen species generation similar to controls, when compared to TGneg T2DM mitochondria. Cav‐3 OE controls and Cav‐3OE T2DM showed increased protein expression of OPA‐1 and Mitofusin 2, compared to TGneg controls and TGneg T2DM. Mitochondrial ultrastructure (i.e., mitochondrial swelling and clustering) was preserved in Cav‐3 OE T2DM hearts compared to TGneg T2DM. Our data suggest that Cav‐3 OE in the heart has the ability to limit injury in the setting of diabetes through regulation of mitochondrial structure and function.

Caveolae are cholesterol and sphingolipid enriched invaginations of the plasma membrane and are considered a subset of lipid rafts. Caveolins (Cav), the structural proteins essential for caveolae formation, are critically involved in isoflurane‐induced protection of the heart. The noble gas helium (He) mimics isoflurane‐induced cardioprotection but the molecular mechanisms is unknown. We hypothesize that He influences Cav signaling in vivo in mice. C57BL/6 mice inhaled either 70% He (n=8) or 30% oxygen (n=8) for 20 min. Thirty min or 24 h after inhalation hearts were excised. Cav‐1/3 expression was determined after sucrose density fractionation of whole heart tissue. A cholesterol assay of whole heart samples was performed. Serum of these animals was tested for Cav‐1/3. He inhalation decreased Cav‐1 (0.4±0.1) and 3 (0.7±0.1, vs con 1.7±0.3 and 1.2±0.2, respectively, p<0.05) expression after 24 h. Buoyant Cav enriched fractions supported the results showing lower Cav‐1 and 3 levels. Cholesterol levels where significantly lower after He inhalation (1.7±0.1 vs con 2.4±0.3). Subcellular fractions showed lower levels of Cav‐1/3 mostly in cytosolic and mitochondrial fractions. In contrast, Cav‐1/3 showed accumulation in serum in He mice 24 h after exposure. These data indicate that Cav‐1 and 3 are secreted into the blood after He inhalation in mice, suggesting circulation factors may be involved in inducing organ protection by He.

Significance Early signaling events leading to protection in the heart under cardiac injury are poorly understood. We identified one such protein, A kinase interacting protein (AKIP1), as a modulator that responds to oxidative stress; up-regulation of AKIP1 showed protection to ischemic injury through enhanced mitochondrial integrity. We show AKIP1 functions as a molecular scaffold via interaction with mitochondrial apoptosis inducing factor and increases protein kinase A activity. These mitochondrial signaling complexes assembled by AKIP1 alter the physiological response of the heart under ischemic stress. Understanding molecular activity and regulation of AKIP1 could lead to novel therapeutic approaches to limit myocardial injury.

We show here that the apposition of plasmamembrane 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.

Copper transporter 1 (CTR1) is the major copper (Cu) influx transporter in mammalian cells. We report here that CTR1 is required for the activation of signaling to the MAPK pathway by the ligands of three major receptor tyrosine kinases (RTK) including FGF, PDGF and EGF. Induction of Erk1/2 phosphorylation was compared in isogenic wild type CTR1(+/+) and CTR1(-/-) cells. Whereas all three ligands increased pErk1/2 in the CTR1(+/+) cells, they failed to do this in CTR1(-/-) cells. While FGF did not enhance the phosphorylation of AKT in the CTR1(+/+) cells, both PDGF and EGF increased pAKT in the CTR1(+/+) but not CTR1(-/-) cells. The deficit in Erk1/2 phosphorylation in the CTR1(-/-) cells was rescued by adding Cu to the medium, and it was induced in CTR1(+/+) cells by treatment with a Cu chelator. Intracellular Cu availability was reduced in the CTR1(-/-) cells as reflected by increased expression of the Cu chaperone CCS. The failure of RTK-induced signaling to both Erk1/2 and AKT suggested the presence of a Cu-dependent step upstream of Ras. The Cu-dependent enzyme SOD1 is responsible for generating the hydrogen peroxide in response to RTK activation that serves to inhibit phosphatases that normally limit RTK signaling. SOD1 activity was reduced by a factor of 17-fold in the CTR1(-/-) cells, and addition of hydrogen peroxide restored signaling. We conclude that Cu acquired from CTR1 is required for signaling in pathways regulated by RTKs that play major roles in development and cancer.

Epicatechin, a dark chocolate flavonol/antioxidant, is linked to cardiovascular cytoprotection. Myocardial infarct size was decreased in epicatechin‐treated mice compared to controls, and this effect was blocked by a DOR antagonist. Low‐dose epicatechin, with little antioxidant activity, is protective; however, the mechanism of this cytoprotection is unknown. We tested if epicatechin (Epi) mediates cardiac protection via modulation of mitochondrial function and membrane structure. Mitochondria were isolated from all experimental groups [saline control, Epi (1mg/kg), naltrindol (Nal, DOR antagonist, 5mg/kg), Epi+Nal, SNC‐121 (OR agonist, 1mg/kg), and SNC‐121+Nal for 10 days] and assayed for oxygen consumption. Compared to saline controls, Epi and SNC‐121 treated mitochondria showed enhanced functional performance through complex I under state III respiration. This effect was attenuated by DOR antagonism with Nal. Further, we studied the effect of Epi on membrane dynamics. The effect of epicatechin on cardiac myocyte membrane dynamics was examined with electron paramagnetic resonance (EPR) spectroscopy. Epi increased membrane order parameter (decreased membrane fluidity) when compared to vehicle treated myocytes, an effect blocked by Nal. In conclusion, epicatechin alters mitochondrial function and membrane fluidity via DOR to produce cardiac protection.