Effect of an oral astaxanthin prodrug (CDX-085) on lipoprotein levels and progression of atherosclerosis in LDLR(-/-) and ApoE(-/-) mice.

UNLABELLED: Oxidative stress and inflammation are key promoters of atherosclerosis and myocardial damage. When orally administered, the novel astaxanthin prodrug CDX-085 delivers high levels of the xanthophyll antioxidant astaxanthin that protects LDL from oxidation and reduces primary thrombosis. In this study, we analyzed whether delivery of astaxanthin from administration of the CDX-085 prodrug reduces plasma lipoprotein levels and the progression of atherosclerosis in low-density lipoprotein receptor negative (LDLR(-/-)) and apolipoprotein E deficient (ApoE(-/-)) mice. METHODS: Relative circulating levels of astaxanthin derived from CDX-085 administration compared to administration of pure astaxanthin was initially evaluated in a canine model. In mouse Study #1, 16 wild-type and 16 LDLR(-/-) mice on 0.5% cholesterol diet supplemented with either 0.0%, 0.08%, 0.2% and 0.4% CDX-085 were used to assess plasma levels and lipoprotein biodistribution measured by FPLC after 4 weeks treatment. In Study #2, 36 male LDLR(-/-) mice were randomized to a 0.5% cholesterol chow diet (CHOW group, n=12) or 0.5% cholesterol chow fortified with 0.08% CDX-085 (n=12) or 0.5% cholesterol chow with 0.4% CDX-085 (n=12) for 12 weeks. In Study #3, 34 male ApoE(-/-) mice were randomized in the same fashion as the Study #2 and fed similar diets for 9 weeks. RESULTS: CDX-085 administration was shown to result in significantly higher levels of circulating astaxanthin (p<0.001 ANOVA) over a 72 h period compared to pure, non-esterified astaxanthin in a single-dose pharmacokinetic study in beagles. In Study #1, plasma astaxanthin levels were 5-9-fold higher in LDLR(-/-) mice compared to wild-type mice. Astaxanthin was highly distributed among all lipoprotein fractions, generally reflecting cholesterol content of lipoproteins. In Study #2, administration of CDX-085 resulted in significantly lower total cholesterol levels (528±68 mg/dL vs. 550±67 mg/dL vs. 602±80 mg/dL, p=0.047) and aortic arch atherosclerosis (9.0±4.2% vs. 9.8±3.5% vs. 13.2±3.6%, p=0.023) in the 0.4% CDX-085 group compared to the 0.08% CDX-085 and CHOW groups, respectively. In ApoE(-/-) mice, a 72% reduction in triglycerides in the 0.4% CDX-085 group and 50% reduction in the 0.08% CDX-085 groups was noted compared to CHOW group (final levels 17±11 mg/dL vs. 30±15 mg/dL vs. 60±32 mg/dL, respectively, p=0.001). CONCLUSION: Oral administration of the novel astaxanthin prodrug CDX-085 shows that it distributes among lipoproteins. CDX-085 lowers total cholesterol and aortic arch atherosclerosis in LDLR(-/-) mice and triglyceride levels in ApoE(-/-) mice and shows promise for further evaluation in human studies.

Novel astaxanthin prodrug (CDX-085) attenuates thrombosis in a mouse model.

BACKGROUND: Cardiovascular disease remains the leading cause of morbidity and premature mortality in most industrialized countries as well as in developing nations. A pro-oxidative state appears to promote and/or exacerbate vascular disease complications. Furthermore, a state of low-grade chronic inflammation can promote increased oxidative stress and lead to endothelial cell and platelet dysfunction ultimately contributing to thrombogenesis. OBJECTIVES: In this study, the effect of a proprietary astaxanthin prodrug (CDX-085) on thrombus formation was investigated using a mouse model of arterial thrombosis. The influence of free astaxanthin, the active drug of CDX-085, on human endothelial cells and rat platelets was evaluated to investigate potential mechanisms of action. METHODS AND RESULTS: Oral administration of CDX-085 (0.4% in chow, approximately 500 mg/kg/day) to 6-8 week old C57BL/6 male mice for 14 days resulted in significant levels of free astaxanthin in the plasma, liver, heart and platelets. When compared to control mice, the CDX-085 fed group exhibited significant increases in basal arterial blood flow and significant delays in occlusive thrombus formation following the onset of vascular endothelial injury. Primary human umbilical vein endothelial cells (HUVECs) and platelets isolated from Wistar-Kyoto rats treated with free astaxanthin demonstrated significantly increased levels of released nitric oxide (NO) and significantly decreased peroxynitrite (ONOO-) levels. CONCLUSION: Observations of increased NO and decreased ONOO- levels in endothelial cells and platelets support a potential mechanism of action for astaxanthin (CDX-085 active drug). These studies support the potential of CDX-085 and its metabolite astaxanthin in the treatment or prevention of thrombotic cardiovascular complications.

Analysis of Connexin43 phosphorylated at S325, S328 and S330 in normoxic and ischemic heart.

The functional consequences of Connexin43 (Cx43) phosphorylation remain largely unexplored. Using an antibody that specifically recognizes Cx43 phosphorylated at serine residues 325, 328 and/or 330 (pS325/328/330-Cx43), we show that labeling of this form of Cx43 as well as of total Cx43 is restricted to the intercalated disk region of normal ventricular tissue. In ischemic heart, significant relocalization of total Cx43 to the lateral edges of myocytes was evident; however pS325/328/330-Cx43 remained predominately at the intercalated disk. Western blots indicated a eightfold decrease in pS325/328/330-Cx43 in ischemic tissue. Peptide-binding- and competition-experiments indicated that our antibody mainly detected Cx43 phosphorylated at S328 and/or S330 in heart tissue. To evaluate how this change in Cx43 phosphorylation contributes to ischemia-induced downregulation of intercellular communication, we stably transfected Cx43(-/-) cells with a Cx43 construct in which serine residues 325, 328 and 330 had been mutated to alanine (Cx43-TM). Cx43-TM was not efficiently processed to isoforms that have been correlated with gap junction assembly. Nevertheless, Cx43-TM cells were electrically coupled, although development of coupling was delayed. Fully opened channels were only rarely observed in Cx43-TM cells, and Lucifer-Yellow-dye-coupling was significantly reduced compared with wild-type cells. These data suggest that phosphorylation of Cx43 at serine residues 325, 328 and/or 330 influences channel permselectivity and regulates the efficiency of gap junction assembly.

Selectivity of connexin 43 channels is regulated through protein kinase C-dependent phosphorylation.

Coordinated contractile activation of the heart and resistance to ischemic injury depend, in part, on the intercellular communication mediated by Cx43-composed gap junctions. The function of these junctions is regulated at multiple levels (assembly to degradation) through phosphorylation at specific sites in the carboxyl terminus (CT) of the Cx43 protein. We show here that the selective permeability of Cx43 junctions is regulated through protein kinase C (PKC)-dependent phosphorylation at serine 368 (S368). Selective permeability was measured in several Cx43-expressing cell lines as the rate constant for intercellular dye diffusion relative to junctional conductance. The selective permeability of Cx43 junctions under control conditions was quite variable, as was the open-state behavior of the comprising channels. Coexpression of the CT of Cx43 as a distinct protein, treatment with a PKC inhibitor, or mutation of S368 to alanine, all reduced (or eliminated) phosphorylation at S368, reduced the incidence of 55- to 70-pS channels, and reduced by 10-fold the selective permeability of the junctions for a small cationic dye. Because PKC activation during preischemic conditioning is cardioprotective during subsequent ischemic episodes, we examined no-flow, ischemic hearts for Cx43 phosphorylated at S368 (pS368). Consistent with early activation of PKC, pS368-Cx43 was increased in ischemic hearts; despite extensive lateralization of total Cx43, pS368-Cx43 remained predominantly at intercalated disks. Our data suggest that the selectivity of gap junction channels at intercalated disks is increased early in ischemia.

Connexins as targets for cancer chemoprevention and chemotherapy.

Cells within a tissue continuously interact to coordinate normal tissue functions and maintain homeostasis. Gap junctional communication (GJC), mediated by the connexin protein family, allows this type of intercellular crosstalk resulting in synchronized and cooperative tissue behavior such as cardiac contraction. In cancer, loss of these types of cell:cell interactions has been shown to facilitate tumorigenesis and enable the autonomous cell behavior associated with transformed cells. Indeed, many human tumor lines demonstrate deficient or aberrant GJC and/or loss of connexin expression. Restoration of exogenous connexin expression/GJC function is correlated with increased cell growth control both in vitro and in vivo. In support of this growth regulatory hypothesis, decreased connexin expression has been observed in situ in early human neoplasia of various organs. Additionally, genetically engineered mice lacking particular connexins (Connexins 32 or 43) exhibit increased susceptibility to radiation and chemically-induced liver and/or lung tumorigenesis. These studies strongly suggest that connexins and GJC serve a tumor suppressor role. Consistent with this proposed role, in a model cell culture system, retinoids and carotenoids up-regulate Connexin43 (Cx43) expression in direct proportion to their ability to suppress carcinogen-induced neoplastic transformation. Here, we discuss the important role of connexins and GJC in tumorigenesis and suggest the possibility of connexins as potential anti-oncogenic targets for chemoprevention and/or chemotherapy.

Temporal regulation of connexin phosphorylation in embryonic and adult tissues.

Gap junctions, composed of proteins from the connexin family, allow for intercellular communication between cells in tissues and are important in development, tissue/cellular homeostasis, and carcinogenesis. Genome databases indicate that there are at least 20 connexins in the mouse and human. Connexin phosphorylation has been implicated in connexin assembly into gap junctions, gap junction turnover, and cell signaling events that occur in response to tumor promoters and oncogenes. Connexin43 (Cx43), the most widely expressed and abundant gap junction protein, can be phosphorylated at several different serine and tyrosine residues. Here, we focus on the dynamic regulation of Cx43 phosphorylation in tissue and how these regulatory events are affected during development, wound healing, and carcinogenesis. The activation of several kinases, including protein kinase A, protein kinase C, p34cdc2/cyclin B kinase, casein kinase 1, mitogen-activated protein kinase, and pp60src kinase, can lead to the phosphorylation of different residues in the C-terminal region of Cx43. The use of antibodies specific for phosphorylation at defined residues has allowed the examination of specific phosphorylation events both in tissue culture and in vivo. These new antibody tools and those under development will allow us to correlate specific phosphorylation events with changes in connexin function.

Altered tumor biology and tumorigenesis in irradiated and chemical carcinogen-treated single and combined connexin32/p27Kip1-deficient mice.

Connexin32 knockout mice (Cx32-KO) exhibit increased chemical and radiation-induced liver and lung tumorigenesis. This increased tumor incidence is associated with altered tumor biology including enhanced tumor progression and an increased percent of MAPK-active tumors. Likewise, mice lacking the tumor suppressor/cell cycle regulator p27Kip1 exhibit increased tumorigenesis in a variety of tissues following chemical and radiation induction. Interestingly, in a double-deficient mouse model (DKO), additional loss of p27Kip1 in a Cx32-KO background results in attenuation of liver and lung tumorigenesis as well as MAPK activation profiles, suggesting pathway interaction. While these mouse strains exhibit altered liver and lung tumor susceptibility following both chemical (DEN) and radiation (X-ray) induction protocols, comparisons of the resulting tumor incidence, multiplicity, tumor progression, and MAPK activation in response to these two distinct carcinogens underscores the separate influence of each individual gene on both tumor formation and activation of specific oncogenic pathways. Furthermore, these studies demonstrate that different carcinogens interact disparately with Cx32/p27Kip1 genotypic backgrounds in situ resulting in varied tumorigenic response.

Deficiency in the gap junction protein connexin32 alters p27Kip1 tumor suppression and MAPK activation in a tissue-specific manner.

Connexin32 knockout mice (Cx32-KO) exhibit increased chemical- and radiation-induced liver and lung tumor formation with many lung tumors demonstrating decreased levels of the tumor suppressor p27KIP1. To determine if p27 deficiency alters Cx32-influenced tumorigenesis, we have generated a Cx32/p27 double-deficient mouse strain (DKO) and show here that exposure of these mice to X-ray radiation resulted in an increase or decrease in tumorigenesis depending on the tissue. Several tissues were highly sensitive to loss of p27 tumor suppressor function (intestine, adrenal, pituitary) resulting in an increased overall tumor burden in DKO mice compared to both wild-type (P<0.005) and Cx32-KO mice (P=0.066). However, additional deletion of p27 in a Cx32-KO background resulted in a statistically significant decrease in the liver tumor incidence suggesting that Cx32 and p27 pathways mechanistically interact. Immunohistochemical analysis revealed an increased percentage of Cx32-KO liver and lung tumors harboring active mitogen-activated protein kinase (Erk1, Erk2) pathways in contrast to lower percentages of activated wild-type (P<0.005) and DKO tumors (P=0.027). Increased MAPK activation in liver tumors did not correlate with Ha-ras codon-61 mutation status. This study demonstrates that tissues dependent on Cx32 tumor suppression, such as the liver and lung, exhibit altered tumorigenesis and tumor biology (MAPK pathway activation) related to p27 status.

The gap junction protein connexin32 is a mouse lung tumor suppressor.

Although loss of connexin expression and/or gap junction intercellular communication correlates with decreased growth control and increased neoplastic potential, there is limited evidence directly linking gap junction intercellular communication function with tumor suppression in situ. Here, we show for the first time that a gap junction protein, connexin32 (Cx32), acts as a lung tumor suppressor in a mouse model. Cx32-deficient nontumorous lung tissue exhibited an increased proliferative index (P < 0.001), and, after exposure to the carcinogen diethylnitrosamine, Cx32-deficient mice exhibited a highly statistically significant (P < 0.001) increase in bronchioloalveolar lung tumor incidence (28 of 45, 62%) and a 45% increase in average multiplicity compared with wild-type mice (7 of 29, 24%). Tumors from Cx32-deficient mice also showed increased activation of mitogen-activated protein kinase (P < 0.001) compared with wild-type tumors, implicating this signaling pathway in Cx32/gap junction intercellular communication-associated lung tumorigenesis.

Mice deficient for the gap junction protein Connexin32 exhibit increased radiation-induced tumorigenesis associated with elevated mitogen-activated protein kinase (p44/Erk1, p42/Erk2) activation.

Loss of connexin expression/gap junction intercellular communication (GJIC) has been correlated with decreased growth control and increased tumorigenesis. Studies utilizing Connexin32 (Cx32)-deficient knockout mice have demonstrated that loss of Cx32 increases susceptibility to chemically induced liver tumorigenesis. Here, in addition to dramatically increased liver tumorigenesis, we show that tumor induction utilizing X-ray radiation resulted in a statistically significant increase in overall tumor burden in Cx32-deficient mice compared with wild-type mice due to tumorigenesis in several other tissues (lung, adrenal, lymph and small intestine) even when excluding prevalent liver tumors. Irradiated Cx32-deficient mice were particularly sensitive to liver tumorigenesis (46% incidence compared with 18% in wild-type mice, P = 0.007) demonstrating that Cx32 functions as a hepatic tumor suppressor in response to radiation-associated mutation events. Cx32-deficient mice also exhibited increased lung tumorigenesis (bronchioloalveolar) with an increased progression to carcinoma when compared with wild-type mice. Two Cx32-deficient mice developed an uncommon, invasive medullary adrenal tumor type (pheochromocytoma) not observed in irradiated wild-type mice. Immunohistochemical analysis revealed increased levels of activated mitogen-activated protein kinase (MAPK) (p44/Erk1, p42/Erk2) in Cx32-deficient mouse liver tumors (P = 0.006), lung tumors (P = 0.056) and adrenal tumors (primary and metastases) compared with wild-type counterparts implicating elevated activation of MAPK-interacting pathways in Cx32-deficient tumorigenesis. Interestingly, lung tumors from Cx32-deficient mice also demonstrated decreased p27Kip1 levels compared with wild-type lung tumors (P = 0.05). This study demonstrates that loss of Cx32/GJIC plays a significant role in radiation-induced tumorigenesis of the liver and importantly that Cx32 may also play a role in tumor suppression and/or tumor progression in other tissue types such as lung and adrenal gland. Additionally, this mouse model suggests that MAPK-related pathways may be preferentially activated or conversely that tumors harboring activated MAPK pathways may selectively progress towards more advanced tumor states in the absence of Cx32-mediated GJIC.

Inducible expression of the gap junction protein connexin43 decreases the neoplastic potential of HT-1080 human fibrosarcoma cells in vitro and in vivo.

Numerous studies have demonstrated a correlation between dysregulation/loss of connexin expression or gap junction intercellular communication (GJIC) function and decreased growth control both in human tumors and tumor cell lines. Likewise, restoration of constitutive connexin expression/function is correlated with increased growth control/decreased tumorigenicity. Here, we show for the first time that inducible restoration of connexin43 (Cx43) expression and GJIC function in a human tumor line of mesenchymal origin (HT-1080, fibrosarcoma) resulted in a lowered neoplastic potential. Specifically, HT-1080 cells induced to express Cx43 demonstrated diminished foci formation when in co-culture with normal fibroblasts, decreased colony formation under anchorage-independent conditions, and reduced tumor growth when injected into immunodeficient mice. These results, obtained utilizing an inducible system that helps address issues of clonal heterogeneity, strongly implicate Cx43 as a tumor suppressor in human tissue of mesenchymal origin and GJIC as a regulatory mechanism for cellular growth control both in vitro and in vivo. This study also further supports the hypothesis that loss of Cx43/GJIC in human tumors may play an important role in the dysregulation of normal growth control.

Somatic cell nuclear transfer in the pig: control of pronuclear formation and integration with improved methods for activation and maintenance of pregnancy.

To clone a pig from somatic cells, we first validated an electrical activation method for use on ovulated oocytes. We then evaluated delayed versus simultaneous activation (DA vs. SA) strategies, the use of 2 nuclear donor cells, and the use of cytoskeletal inhibitors during nuclear transfer. Using enucleated ovulated oocytes as cytoplasts for fetal fibroblast nuclei and transferring cloned embryos into a recipient within 2 h of activation, a 2-h delay between electrical fusion and activation yielded blastocysts more reliably and with a higher nuclear count than did SA. Comparable rates of development using DA were obtained following culture of embryos cloned from ovulated or in vitro-matured cytoplasts and fibroblast or cumulus nuclei. Treatment of cloned embryos with cytochalasin B (CB) postfusion and for 6 h after DA had no impact on blastocyst development as compared with CB treatment postfusion only. Inclusion of a microtubule inhibitor such as nocodozole with CB before and after DA improved nuclear retention and favored the formation of single pronuclei in experiments using a membrane dye to reliably monitor fusion. However, no improvement in blastocyst development was observed. Using fetal fibroblasts as nuclear donor cells, a live cloned piglet was produced in a pregnancy that was maintained by cotransfer of parthenogenetic embryos.