Role of ovarian cancer antigen CA125/MUC16 in development and cancer

Cancer antigen 125 (CA125) is a tumor antigen that is routinely used to monitor the disease progress and the outcome of treatment in ovarian cancer patients. Elevated serum levels of CA125 are detected in over 80% of epithelial ovarian cancer patients. CA125 is a high molecular weight (>1M Dalton) mucin-type glycoprotein encoded by the MUC16 gene on human chromosome 19. Although MUC16 has served as the best serum marker for monitoring growth of ovarian cancer, roles for MUC16 in normal physiology and ovarian cancer are largely unknown. To understand the biological functions of MUC16, I characterized a mouse Muc16 homolog on chromosome 9 by means of expression pattern profiling, phenotype analysis of Muc16 knockout mice, and in vitro and in vivo studies of Muc16 null transformed ovarian surface epithelial (OSE) cells. ^ The mouse Muc16 homolog shares a conserved genomic structure with human MUC16. In addition to being expressed in mouse ovarian cancer, mouse Muc16 mRNA and protein were expressed in the mesothelia covering the heart, lung, ovary, oviduct, spleen, testis, and uterus. The conserved genomic structure and expression pattern of mouse Muc16 to human MUC16 suggests that mouse Muc16 is the ortholog of human MUC16. To understand the biological functions of Muc16, I generated Muc16 knockout mice. Muc16 knockout mice were viable, fertile and normal by one year of age. However, between 18 and 24 months of age, Muc16 knockout mice developed various tissue abnormalities such as ovarian cysts and tumors of the liver and other peritoneal organs. To determine the role of MUC16 in ovarian cancer progression, I established Muc16 null transformed ovarian surface epithelial (OSE) cell lines, following the same method to develop mouse model of epithelial ovarian cancer (Orsulic et al., 2002). Loss of Muc16 did not affect cell morphology, cell proliferation rate, or tumorigenic potential. However, Muc16-null OSE cells showed decreased attachment to extracellular matrix proteins as well as to primary mouse peritoneal mesothelial cells. Peritoneal mesothelia are the most frequent implantation sites of ovarian cancer. Furthermore, a pilot transplantation assay suggests that Muc16 null transformed OSE cells formed less disseminated tumors in the peritoneal cavity compared to wild-type OSE cells. ^ In conclusion, these results demonstrate that MUC16 is not required for normal mouse development or reproduction, but plays important roles in tissue homeostasis, ovarian cancer cell adhesion and dissemination. This study provides the first in vivo evidence of the roles of MUC16 in development, as well as ovarian cancer progression and dissemination. These studies offer valuable insights into possible mechanisms of ovarian cancer development and potential molecular targets for ovarian cancer treatment. ^

Elucidating the IGFBP2 signaling pathway in glioma development and progression

Diffuse gliomas are highly lethal central nervous system malignancies which, unfortunately, are the most common primary brain tumor and also the least responsive to the very few therapeutic modalities currently available to treat them. IGFBP2 is a newly recognized oncogene that is operative in multiple cancer types, including glioma, and shows promise for a targeted therapeutic approach. Elevated IGFBP2 expression is present in high-grade glioma and correlates with poor survival. We have previously demonstrated that IGFBP2 induces glioma development and progression in a spontaneous glioma mouse model, which highlighted its significance and potential for future therapy. However, we did not yet know the key physiological pathways associated with this newly characterized oncogene. We first evaluated human glioma genomics data harnessed from the publicly available Rembrandt source to identify major pathways associated with IGFBP2 expression. Integrin and ILK, among other cell migration and invasion-related pathways, were the most prominently associated. We confirmed that these pathways are regulated by IGFBP2 in glioma cells lines, and demonstrated that 1) IGFBP2 activates integrin α5β1, leading to the activation of key pathways important in glioma; 2) IGFBP2 mediates cell migration pathways through ILK; and 3) IGFBP2 activates NF-kB via an integrin α5 interaction. We then sought to determine whether this was a physiologically active signaling pathway in vivo by assessing its ability to induce glioma progression in the RCAS/tv-a spontaneous glioma mouse model. We found that ILK is a key downstream mediator of IGFBP2 that is required for the induction of glioma progression. Most significantly, a genetic therapeutic approach revealed that perturbation of any point in the pathway thwarted tumor progression, providing strong evidence that targeting the key players could potentially produce a significant benefit for human glioma patients. The elucidation of this signaling pathway is a critical step, since efforts to create a small molecule drug targeting IGFBP2 have so far not been successful, but a number of inhibitors of the other pathway constituents, including ILK, integrin and NF-kB, have been developed.

Breast cancer risk with aromatase inhibition and phytoestrogen-free diet

In this thesis a mouse model was used to examine the effect of pubertal estrogen inhibition and a phytoestrogen-free diet on the development of mammary glands. The study question was does treatment with aromatase inhibitor during puberty increase susceptibility to breast cancer among cohorts that consumed a diet free of phytoestrogens. The study design consisted of a cohort of mice treated with aromatase inhibitor, letrozole, during puberty and a vehicular group that was used as a control. Both groups were fed a diet free of phytoestrogens from the time of weaning until sacrifice during adulthood. The study aimed to assess mammary gland development in terms of breast cancer risk. The methods employed in this research included morphological and histological analysis of mammary glands, as well as estradiol, RNA and protein analysis. The main finding of the study was that mice exposed to aromatase inhibitor during puberty developed mammary glands with specific characteristics suggestive of vulnerability to oncogenesis such as increased lateral branching, increased number of glands, increase ductal hyperplasia, and diminished expression of TGFβ and p27 protein levels. The conclusions suggest that puberty is a critical period in which the mammary gland is susceptible to environmental threats that may result in deleterious epigenetic effects leading to an increased breast cancer risk in adulthood. This study has several public health implications; the most significant is that environmental threats during puberty may result in adverse mammary gland development and that phytoestrogen sources in the diet are necessary for normal maturation of the mammary glands.^

DIRECT EFFECTS OF METFORMIN ON PI3K AND RAS SIGNALING IN ENDOMETRIAL CANCER

Metformin has antiproliferative effects through the activation of AMPK and has gained interest as an antineoplastic agent in several cancer types, although studies in endometrial cancer (EC) are limited. The aims of this project were to evaluate pathways targeted by metformin in EC, investigate mechanisms by which metformin exerts its antiproliferative effects, and explore rational combination therapies with other targeted agents. Three EC cell lines were used to evaluate metformin’s effect on cell proliferation, PI3K and Ras-MAPK signaling, and apoptosis. A xenograft mouse model was also used to evaluate the effects of metformin treatment on in vivo tumor growth. These preliminary studies demonstrated that K-Ras mutant cell lines exhibited a decreased proliferative rate, reduced tumor growth, and increased apoptosis in response to metformin compared to K-Ras wild-type cells. To test the hypothesis that mutant K-Ras may predict response to metformin, murine EC cells with loss of PTEN and expressing mutant K-RasG12D were transfected to re-express PTEN or have K-Ras silenced using siRNA. While PTEN expression did not alter response to metformin, cells in which K-Ras was silenced displayed reduced sensitivity to metformin. Mislocalization of K-Ras to the cytoplasm is associated with decreased signaling and induction of apoptosis. Metformin’s effect on K-Ras localization was analyzed by confocal microscopy in cells expressing oncogenic GFP-K-RasG12V. Metformin demonstrated concentration-dependent mislocalization of K-Ras to the cytoplasm. Mislocalization of K-Ras to the cytoplasm was confirmed in K-Ras mutant EC cells (Hec1A) by cell fractionation in response to metformin 1 and 5 mM (p=0.008 and p=0.004). This effect appears to be AMPK-independent as combined treatment with Compound C, an AMPK inhibitor, did not alter K-Ras localization. Furthermore, treatment of EC cells with metformin in combination with PI3K inhibitors resulted in a significant decrease in proliferation than either agent or metformin alone. While metformin exerts antineoplastic effects by activation of AMPK and decreased PI3K signaling, our data suggest that metformin may also disrupt localization of K-Ras and hence its signaling in an AMPK-independent manner. This has important implications in defining patients who may benefit from metformin in combination with other targeted agents, such as mTOR inhibitors.

Chronic stress promotes tumor growth through increased BDNF production and neo-innervation

Background: Activation of the sympathetic nervous system (SNS) in response to chronic biobehavioral stress results in high levels of catecholamines and persistent activation of adrenergic signaling, which promotes tumor growth and progression. However it is unknown how catecholamine levels within the tumor exceed systemic levels in circulation. I hypothesized that neo-innervation of tumors is required for stress-mediated effects on tumor growth. Results: First, I examined whether sympathetic nerves are present in human ovarian cancer samples as well as orthotopic ovarian cancer models. Immunohistochemical (IHC) staining for neurofilament revealed that catecholaminergic neurons are present within tumor tissue. In order to determine whether chronic stress affects the density of nerves in the tumor, I utilized an orthotopic mouse model of ovarian cancer that was exposed to daily restraint stress. IHC analysis revealed that nerve density in tumors increased by more than three-fold in stressed animals versus non-stressed controls. IHC analysis suggested that this results from both recruitment of existing neurons (axonogenesis) as well as new neuron formation (neurogenesis) within the tumor. To determine how tumors are recruiting nerve growth, I utilized a PCR array analysis of 84 nerve growth related genes and their receptors, which showed that stimulation of the SKOV3 ovarian cancer cell line with norepinephrine (NE) leads to increased expression of several neurotrophins, including brain-derived neurotrophic factor (BDNF). Neurite extension assays showed that media conditioned by ovarian cancer cell lines is capable of inducing neurite outgrowth in differentiated neuron-like PC12 cells, and NE treatment of cancer cells potentiates this effect. Norepinephrine-induced neurite extension was abolished after BDNF silencing by siRNA, suggesting that BDNF is critical to tumor cell-induced nerve growth. in vivo BDNF inhibition resulted in complete abrogation of stress-induced increases in tumor weight and nerve density, as well as downstream markers of stress. Discussion: These studies indicate that adrenergic signalling induced by chronic stress promotes neo-innervation in the tumor microenvironment. This results in a mutually beneficial relationship between the tumor cells and neurons. This work is crucial for providing a link between chronic stress and its effects on the tumor and its microenvironment. The data shown here aims to open new venues that can be used in development of therapies designed to block the stress effects on tumor growth.

THE NEW ROLE OF PROPROTEIN CONVERTASE SUBTILISIN/KEXIN TYPE 9: A CONNECTION OF PROPROTEIN CONVERTASE SUBTILISIN/KEXIN TYPE 9, APOLIPOPROTEIN B, and AUTOPHAGY

Plasma low-density lipoprotein (LDL) levels are positively correlated with the incidence of coronary artery disease. In the circulation, the plasma LDL clearance is mainly achieved by the uptake via LDL receptor (LDLR). Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a newly discovered gene, playing an important role in LDL metabolism. Gain-of-function mutations of PCSK9 lead to hypercholesterolemia and loss-of-function mutations of PCSK9 are associated with decrease of LDL cholesterol. The effects of PCSK9 on cholesterol levels are the consequence of a strong interaction between the catalytic domain of PCSK9 and epidermal growth factor-like repeat A (EGF-A) domain of LDLR on the cell surface of hepatocytes. This PCSK9/LDLR complex enters the cell via endocytosis, where both PCSK9 and LDLR are removed via the lysosome pathway, resulting in decreased levels of LDLR and accumulation of LDL in the plasma. However, whether this is the exclusive function of PCSK9 on LDL metabolism was challenged by us; we observed PCSK9 interacted with apolipoprotein B (apoB) and increased apoB production, irrespective of the LDLR. ApoB is the primary structure protein of LDL particle and it also serves as the ligand for the LDL receptor. There is ample evidence showing that the levels of apoB are a better indicator for heart disease than either total cholesterol or LDL cholesterol levels. We used a second-generation adenoviral vector to overexpress PCSK9 (Ad-PCSK9) in wild-type C57BL/6 and LDLR deficient mice (Ldlr-/- and Ldlr-/-Apobec1-/-). Our study revealed that overexpression of PCSK9 promoted the production and secretion of apoB in the form of very-low density lipoprotein (VLDL), which is the precursor of LDL, in the 3 mouse models studied (C57BL/6J, Ldlr-/-, and Ldlr-/-Apobec1-/-). The increased apoB production in mice was regulated at post-transcriptional levels, since there was no difference in apoB mRNA levels between mice treated with Ad-PCSK9 and control vector Ad-Null. By using pulse-chase experiment on primary hepatocytes, we showed that overexpression of PCSK9 increased the secretion of apoB, independent of LDLR. In the circulation, we showed that PCSK9 was associated with LDL particles. By using 3 different protein–protein interaction assays of co-immunoprecipitation, mammalian two-hybrid system, and in situ proximity ligation assay, we demonstrated a direct protein–protein interaction between PCSK9 and apoB. The impact of this interaction inhibited the physiological removal process of apoB via autophagosome/lysosome pathway in an LDLR-independent fashion, resulting in increased production and secretion of apoB-containing lipoproteins. The significance of this process was shown in the Pcsk9 knockout mice in the background of Ldlr-/-Apobec1-/- mice (triple knockout mice); in the absence of Pcsk9 (triple knockout mice) the levels of cholesterol, triacylglycerol, and apoB decreased significantly in comparison to that of Ldlr-/-Apobec1-/- mice. Taken together, our study demonstrated a direct intracellular interaction of PCSK9 with apoB, resulting in the inhibition of apoB degradation via the autophagosome/lysosome pathway independent of LDLR. This discovery provides a new concept of the importance of PCSK9 and suggests new approaches for the therapeutic intervention of hyperlipidemia.

ROLE OF STAT3 IN KERATINOCYTE STEM CELLS DURING SKIN TUMORIGENESIS

STATs play crucial roles in a wide variety of biological functions, including development, proliferation, differentiation, migration and in cancer development. In the present study, we examined the impact of Stat3 deletion or activation on behavior of keratinocytes, including keratinocyte stem cells (KSCs). Deletion of Stat3 specifically in the bulge region of the hair follicle using K15.CrePR1 X Stat3fl/fl mice led to decreased tumor development by altering survival of bulge region KSCs. To further understand the role of KSCs in skin tumorigenesis, K5.Stat3C transgenic (Tg) mice which express a constitutively active/dimerized form of Stat3 called Stat3C via the bovine keratin 5 (K5) promoter were studied. The number of CD34 and α6 integrin positive cells was significantly reduced in Tg mice as compared to non-transgenic (NTg) littermates. There was a concomitant increase in the progenitor populations (Lgr-6, Lrig-1 and Sca-1) in the Tg mice vs. the stem cell population (CD34 and Keratin15). To investigate the mechanism underlying the increase in the progenitor population at the expense of bulge region KSCs we examined if Stat3C expression was involved in inducing migration of the bulge region KSCs. There was altered β-catenin and α6-integrin expression in the hair follicles of Tg mice, which may have contributed to reduced adhesive interactions between the epithelial cells and the basement membrane facilitating migration out of the niche. To further study the effect of Stat3 on differentiation of keratinocytes we analyzed the epidermal keratinocytes in K5.Cre X Stat3fl/fl mice. There was an increase in the expression of epidermal differentiation markers in the Stat3 knockout mice. These data suggest that deletion of Stat3 in the epidermis and hair follicle induced differentiation in these cells. Preliminary studies done with the BK5.Stat3C mouse model suggests that multiple hair follicle stem/progenitor populations may be involved in skin tumor development and progression in this model of skin tumorigenesis. Overall, these data suggest that Stat3 plays an important role in differentiation as well as migration of keratinocytes and that these effects may play a role during epithelial carcinogenesis.

Molecular Mechanisms of Vascular Disease in Patients with Rare Variants in MYH11

Thoracic aortic aneurysms and dissections (TAAD) are the primary disease affecting the thoracic ascending aorta, with an incidence rate of 10.4/100,000. Although about 20% of patients carry a mutation in a single gene that causes their disease, the remaining 80% of patients may also have genetic factors that increase their risk for developing TAAD. Many of the genes that predispose to TAAD encode proteins involved in smooth muscle cell (SMC) contraction and the disease-causing mutations are predicted to disrupt contractile function. SMCs are the predominant cell type in the ascending aortic wall. Mutations in MYH11, encoding the smooth muscle specific myosin heavy chain, are a rare cause of inherited TAAD. However, rare but recurrent non-synonymous variants in MYH11 are present in the general population but do not cause inherited TAAD. The goal of this study was to assess the potential role of these rare variants in vascular diseases. Two distinct variants were selected: the most commonly seen rare variant, MYH11 R247C, and a duplication of the chromosomal region spanning the MYH11 locus at 16p13.1. Genetic analyses indicated that both of these variants were significantly enriched in patients with TAAD compared with controls. A knock-in mouse model of the Myh11 R247C rare variant was generated, and these mice survive and reproduce normally. They have no structural abnormalities of the aorta or signs of aortic disease, but do have decreased aortic contractility. Myh11R247C/R247C mice also have increased proliferative response to vascular injury in vivo and increased proliferation of SMCs in vitro. Myh11R247C/R247C SMCs have decreased contractile gene and protein expression and are dedifferentiated. In fibroblasts, myosin force generation is required for maturation of focal adhesions, and enhancers of RhoA activity replace enhancers of Rac1 activity as maturation occurs. Consistent with these previous findings, focal adhesions are smaller in Myh11R247C/R247C SMCs, and there is decreased RhoA activation. A RhoA activator (CN03) rescues the dedifferentiated phenotype of Myh11R247C/R247C SMCs. Myh11R247C/R247C mice were bred with an existing murine model of aneurysm formation, the Acta2-/- mouse. Over time, mice carrying the R247C allele in conjunction with heterozygous or homozygous loss of Acta2 had significantly increased aortic diameter, and a more rapid accumulation of pathologic markers. These results suggest that the Myh11 R247C rare variant acts as a modifier gene increasing the risk for and severity of TAAD in mice. In patients with 16p13.1 duplications, aortic MYH11 expression is increased, but there is no corresponding increase in smooth muscle myosin heavy chain protein. Using SMCs that overexpress Myh11, we identified alterations in SMC phenotype leading to excessive protein turnover. All contractile proteins, not just myosin, are affected, and the proteins are turned over by autophagic degradation. Surprisingly, these cells are also more contractile compared with wild-type SMCs. The results described in this dissertation firmly establish that rare variants in MYH11 significantly affect the phenotype of SMCs. Further, the data suggests that these rare variants do increase the risk of TAAD via pathways involving altered SMC phenotype and contraction. Therefore, this study validates that these rare genetic variants alter vascular SMCs and provides model systems to explore the contribution of rare variants to disease.

CHARACTERIZATION OF TCL1-Tg:P53-/- MICE THAT RESEMBLE HUMAN CHRONIC LYMPHOCYTIC LEUKEMIA WITH 17P-DELETION

Chronic lymphocytic leukemia (CLL) is the most common adult leukemia in the United Statesand Europe. CLL patients with deletion of chromosome 17p, where the tumor suppressor p53 gene is located, often develop a more aggressive disease with poor clinical outcomes. However, the underlying mechanism remains unclear. In order to understand the underneath mechanism in vivo, I have recently generated mice with Eu-TCL1-Tg:p53-/- genotype and showed that these mice develop aggressive leukemia that resembles human CLL with 17p deletion. The Eu-TCL1-Tg:p53-/- mice developed CLL disease at 3-4 months, significantly earlier than the parental Eu-TCL1-Tg mice that developed CLL disease at 8-12 months. Flow cytometry analysis showed that the CD5+/ IgM+ cell population appeared in the peritoneal cavity, bone marrow, and the spleens of Eu-TCL1-Tg:p53-/- mice significantly earlier than that of the parental Eu-TCL1-Tg mice. Massive infiltration and accumulation of leukemia cells were found in the spleen and peritoneal cavity. In vitro study showed that the leukemia cells isolated from the Eu-TCL1-Tg:p53-/- mice were more resistant to fludarabine treatment than the leukemia cells isolated from spleens of Eu-TCL1-Tg mice. Interestingly, TUNEL assay revealed that there was higher apoptotic cell death found in the Eu-TCL1-Tg spleen tissue compared to the spleens of the Eu-TCL1-Tg:p53-/- mice, suggesting that the loss of p53 compromises the apoptotic process in vivo, and this might in part explain the drug resistant phenotype of CLL cells with 17p-deletion. In the present study, we further demonstrated that the p53 deficiency in the TCL1 transgenic mice resulted in significant down-regulation of microRNAs miR-15a and miR16-1, associated with a substantial up-regulation of Mcl-1, suggesting that the p53-miR15a/16-Mcl-1 axis may play an important role in CLL pathogenesis. Interestingly, we also found that loss of p53 resulted in a significant decrease in expression of the miR-30 family especially miR-30d in leukemia lymphocytes from the Eu-TCL1-Tg:p53-/- mice. Such down-regulation of those microRNAs and up-regulation of Mcl-1 were also found in primary leukemia cells from CLL patients with 17p deletion. To further exam the biological significance of decrease in the miR-30 family in CLL, we investigated the potential involvement of EZH2 (enhancer of zeste homolog 2), a component of the Polycomb repressive complex known to be a downstream target of miR-30d and plays a role in disease progression in several solid cancers. RT-PCR and western blot analyses showed that both EZH2 mRNA transcript and protein levels were significantly increased in the lymphocytes of Eu-TCL1-Tg:p53-/- mice relative to Eu-TCL1-Tg mice. Exposure of leukemia cells isolated from Eu-TCL1-Tg:p53-/- mice to the EZH2 inhibitor 3-deazaneplanocin (DZNep) led to induction of apoptosis, suggesting EZH2 may play a role in promoting CLL cell survival and this may contribute to the aggressive phenotype of CLL with loss of p53. Our study has created a novel CLL mouse model, and suggests that the p53/miR15a/16-Mcl-1 axis & p53/miR30d-EZH2 may contribute to the aggressive phenotype and drug resistance in CLL cells with loss of p53.

INTERACTION OF BACILLUS ANTHRACIS EXOSPORIUM PROTEIN BCLA WITH COMPLEMENT FACTOR H AND SPORE PERSISTENCE IN THE LUNG

Anthrax outbreaks in the United States and Europe and its potential use as a bioweapon have made Bacillus anthracis an interest of study. Anthrax infections are caused by the entry of B. anthracis spores into the host via the respiratory system, the gastrointestinal tract, cuts or wounds in the skin, and injection. Among these four forms, inhalational anthrax has the highest lethality rate and persistence of spores in the lungs of animals following pulmonary exposure has been noted for decades. However, details or mechanisms of spore persistence were not known. In this study, we investigated spore persistence in a mouse model. The results suggest that B. anthracis spores have special properties that promote persistence in the lung, and that there may be multiple mechanisms contributing to spore persistence. Moreover, recent discoveries from our laboratory suggest that spores evolved a sophisticated mechanism to interact with the host complement system. The complement system is a crucial part of the host defense mechanism against foreign microorganisms. Knowledge of the specific interactions that occur between the complement system and B. anthracis was limited. Studies performed in our laboratory have suggested that spores of B. anthracis can target specific proteins, such as Factor H (fH) of the complement system. Spores of B. anthracis are enclosed by an exosporium, which consists of a basal layer surrounded by a nap of hair-like filaments. The major structural component of the filaments is called Bacillus collagen-like protein of anthracis (BclA), which comprises a central collagen-like region and a globular C-terminal domain. BclA is the first point of contact with the innate system of an infected host. In this study, we investigated the molecular details of BclA-fH interaction with respect to the specific binding mechanism and the functional significance of this interaction in a murine model of anthrax infection. We hypothesized that the recruitment of fH to the spore surface by BclA limits the extent of complement activation and promotes pathogen survival and persistence in the infected host. Findings from this study are significant to understanding how to treat post-exposure prophylaxis and improve our knowledge of spores with the host immune system.

Delayed Thrombus Resolution and Fibroproliferative Vascular Wound Healing From Deficiency of Type III Collagen: a Paradoxical Mechanism for Tissue Fragility

Vascular Ehlers-Danlos syndrome is a heritable disease of connective tissue caused by mutations in COL3A1, conferring a tissue deficiency of type III collagen. Cutaneous wounds heal poorly in these patients, and they are susceptible to spontaneous and catastrophic rupture of expansible hollow organs like the gut, uterus, and medium-sized to large arteries, which leads to premature death. Although the predisposition for organ rupture is often attributed to inherent tissue fragility, investigation of arteries from a haploinsufficient Col3a1 mouse model (Col3a1+/-) demonstrates that mutant arteries withstand even supraphysiologic pressures comparably to wild-type vessels. We hypothesize that injury that elicits occlusive thrombi instead unmasks defective thrombus resolution resulting from impaired production of type III collagen, which causes deranged remodeling of matrix, persistent inflammation, and dysregulated behavior by resident myofibroblasts, culminating in the development of penetrating neovascular channels that disrupt the mechanical integrity of the arterial wall. Vascular injury and thrombus formation following ligation of the carotid artery reveals an abnormal persistence and elevated burden of occlusive thrombi at 21 post-operative days in vessels from Col3a1+/- mice, as opposed to near complete resolution and formation of a patent and mature neointima in wild-type mice. At only 14 days, both groups harbor comparable burdens of resolving thrombi, but wild-type mice increase production of type III collagen in actively resolving tissues, while mutant mice do not. Rather, thrombi in mutant mice contain higher burdens of macrophages and proliferative myofibroblasts, which persist through 21 days while wild-type thrombi, inflammatory cells, and proliferation all regress. At the same time that increased macrophage burdens were observed at 14 and 21 days post ligation, the medial layer of mutant arterial walls concurrently harbored a significantly higher incidence of penetrating neovessels compared with those in wild-type mice. To assess whether limited type III collagen production alters myofibroblast behavior, fibroblasts from vEDS patients with COL3A1 missense mutations were seeded into three-dimensional fibrin gel constructs and stimulated with transforming growth factor-β1 to initiate myofibroblast differentiation. Although early signaling events occur similarly in all cell lines, late extracellular matrix- and mechanically-regulated events like transcriptional upregulation of type I and type III collagen secretion are delayed in mutant cultures, while transcription of genes encoding intracellular contractile machinery is increased. Sophisticated imaging of collagen synthesized de novo by resident myofibroblasts visualizes complex matrix reorganization by control cells but only meager remodeling by COL3A1 mutant cells, concordant with their compensatory contraction to maintain tension in the matrix. Finally, administration of immunosuppressive rapamycin to mice following carotid ligation sufficiently halts the initial inflammatory phase of thrombus resolution and fully prevents both myofibroblast migration into the thrombus and the differential development of neovessels between mutant and wild-type mice, suggesting that pathological defects in mutant arteries develop secondarily to myofibroblast dysfunction and chronic inflammatory stimulation, rather than as a manifestation of tissue fragility. Together these data establish evidence that pathological defects in the vessel wall architecture develop in mutant arteries as sequelae to abnormal healing and remodeling responses activated by arterial injury. Thus, these data support the hypothesis that events threatening the integrity of type III collagen-deficient vessels develop not as a result of inherent tissue weakness and fragility at baseline but instead as an episodic byproduct of abnormally persistent granulation tissue and fibroproliferative intravascular remodeling.

Regulation of apoptosis during the pathogenesis of non -melanoma skin cancer

Previous studies from our lab have shown distinctive patterns of expression of bcl-2 gene family members in human nonmelanoma skin cancer (NMSC). To further evaluate the significance of these observations and to study the effects of cell death deregulation during skin carcinogenesis, we generated a transgenic mouse model (HK1.bcl-2) using the human keratin 1 promoter to target the expression of a human bcl-2 minigene to the epidermis. Transgenic protein expression was confirmed in all the layers of the epidermis except the stratum corneum using immunohistochemistry. Multifocal epidermal hyperplasia, without associated hyperkeratosis, was observed in newborn HK1.bcl-2 mice. Immunofluorescence staining using monoclonal antibodies specific for a variety of differentiation markers revealed aberrant expression of keratin 6 (K6) in the transgenic epidermis. Epidermal proliferative indexes, assessed by anti-BrdUrd immunofluorescence staining, were similar in control and transgenic newborn mice, but suprabasal proliferating cells were seen within the hyperplastic areas of the transgenic mouse skin. Spontaneous apoptotic indices of the epidermis were similar in both control and HK1.bcl-2 transgenic newborn mice, however, after UV-B irradiation, the number of "sunburn cells" was significantly higher in the control compared to the HK1.bcl-2 transgenic animals.^ Adult HK1.bcl-2 and control littermate mice were used in UV-B and chemical carcinogenesis protocols including DMBA + TPA. UV-B irradiated control and HK1.bcl-2 mice had comparable incidence of tumors than the controls, but the mean latency period was significantly shorter in the HK1.bcl-2 transgenic. Both control and transgenic animals included in chemical carcinogenesis protocols required application of both the initiating (DMBA) and promoting (TPA) agents to develop tumors. The frequency, number, and latency of tumor formation was similar in both groups of animals, however, HK1.bcl-2 mice exhibited a rate of conversion from benign papilloma to carcinoma 2.5 times greater than controls.^ Similar carcinogenesis experiments were performed using newborn mice. HK1.bcl-2 mice treated with UV-B plus TPA have a three fold greater incidence of tumor formation compared to controls littermates. HK1.bcl-2 transgenic animals also exhibited a shorter latency for papilloma formation when treated with DMBA plus TPA.^ HK1.bcl-2/v-Ha-ras double transgenic mice shared phenotypic features of both HK1.v-Ha-ras and HK1.bcl-2 transgenic mice, and exhibited focal areas of augmented hyperplasia. These double transgenic mice were susceptible to tumor formation by treatment with TPA alone.^ Cultures of primary keratinocytes were established from control, HK1.bcl-2, HK1.Ha-ras, and HK1.bcl-2/v-Ha-ras newborn mice. Cell viability was determined after exposure of the cells to UV-B irradiation, DMBA, TPA, or TGF-$\beta$1. Internucleosomal DNA fragmentation ("ladders") and morphological cellular changes compatible with apoptotic cell death were observed after the application of all these agents. HK1.bcl-2 keratinocytes were resistant to cell death induction by all of these agents except TGF-$\beta$1. HK1.Ha-ras cells had a higher spontaneous rate of cell death which could be compensated by co-expression of bcl-2.^ These findings suggest that bcl-2 dependent cell death suppression may be an important component of multistep skin carcinogenesis. ^

Regulations of SOX9 activity during chondrogenesis

Sox9 is a transcription factor required for chondrocyte differentiation and cartilage formation. In an effort to identify SOX9 interacting protein(s), we screened a chondrocyte cDNA library with a modified yeast two-hybrid method, Son of Sevenless (SOS) recruitment system (SRS). The catalytic subunit of cyclic AMP-dependent protein kinase A (PKA-Cα) and a new long form of c-Maf transcription factor (Lc-Maf) were found to interact specifically with SOX9. We showed here that two PKA phosphorylation consensus sites of SOX9 could be phosphorylated by PKA in vitro as well as in vivo. PKA phosphorylation of SOX9 increases its DNA binding and transcriptional activities on a Col2a1 chondrocyte-specific enhancer. Mutations of these two PKA phosphorylation sites markedly decreased the activation of SOX9 by PKA. ^ To test whether parathyroid hormone-related peptide (PTHrP) signaling results in SOX9 phosphorylation, we generated a phosphospecific antibody that specifically recognizes SOX9 that is phosphorylated at serine 181 (S 181) one of the two consensus PKA phosphorylation sites. Addition of PTHrP to COS7 cells cotransfected with SOX9 and PTH/PTHrP receptor strongly increased phosphorylation of SOX9 at S181; this phosphorylation was blocked by a PKA-specific inhibitor. In similar experiments we showed that PTHrP increased the activity of a SOX9-dependent Col2a1 enhancer. This increase in activity was abolished when a SOX9 mutant was used containing serine-to-alanine substitution in the two consensus PKA phosphorylation sites of SOX9. Using our phosphospecific SOX9 antibody we showed by immunohistochemistry of mouse embryos that Sox9 phosphorylated at S181 was localized almost exclusively in the pre-hypertrophic zone of the growth plate, an area corresponding to the major site of expression of PTH/PTHrP receptor. In contrast, no phosphorylation of Sox9 at S181 was detected in growth plates of PTH/PTHrP receptor null mutant mice. Sox9, regardless of phosphorylation state, was present in all chondrocytes of both genotypes except in hypertrophic chondrocytes. Thus, Sox9 is a target of PTHrP signaling and the PTHrP-dependent phosphorylation of SOX9 enhances its transcriptional activity. ^ In order to investigate the in vivo function of Sox9 phosphorylation by PKA, we are generating a mouse model of mutant Sox9 harboring point mutations in two PKA phosphorylation sites. Preliminary results indicated that heterozygous mice containing half amount of mutant Sox9 that can not be phosphorylated by PKA have normal skeletal phenotype and homozygous mice are being generated. ^ Lc-Maf encodes an extra ten amino acids at the carboxyl terminus of c-Maf and contains a completely different 3′ untranslated region. The interaction between SOX9 and Lc-Maf was further confirmed by co-immunoprecipitation and GST-pull down assays, which mapped the interacting domains of SOX9 to HMG DNA binding domain and that of Lc-Maf to basic leusine zipper motif. In situ hybridizations showed that RNA of Lc-Maf coexpressed with those of Sox9 and Col2a1 in areas of mesenchymal condensation during the early stages of mouse embryo development. A DNA binding site of Lc-Maf was identified at the 5′ part of a 48-bp Col2a1 enhancer element near the HMG binding site of SOX9. Lc-Maf and SOX9 synergistically activated a luciferase reporter plasmid containing a Col2al enhancer and increased the transcription of endogenous Col2a1 gene. In summary, Lc-Maf is the first identified SOX9-interating protein during chondrogenesis and may be an important activator of Col2a1 gene. ^

Estudio Multimodal de la Funcionalidad Cortical en ratones Transgénicos con Síndrome X Frágil y la modulación del dolor

El dolor es un síntoma frecuente en la práctica médica. En España, un estudio realizado en el año 2000 demostró que cada médico atiende un promedio de 181 pacientes con dolor por mes, la mayoría de ellos con dolor crónico moderado1. Del 7%-8% de la población europea está afectada y hasta el 5% puede ser grave2-3, se estima, que afecta a más de dos millones de españoles4. En la consulta de Atención Primaria, los pacientes con dolor neuropático tienen tasas de depresión mucho mayores 5-6-7. El dolor neuropático8 es el dolor causado por daño o enfermedad que afecta al sistema somato-sensorial, es un problema de salud pública con un alto coste laboral, debido a que existe cierto desconocimiento de sus singularidades, tanto de su diagnóstico como de su tratamiento, que al fallar, el dolor se perpetúa y se hace más rebelde a la hora de tratarlo, en la mayoría de las ocasiones pasa a ser crónico. Los mecanismos fisiopatológicos son evolutivos, se trata de un proceso progresivo e integrado que avanza si no recibe tratamiento, ocasionando graves repercusiones en la calidad de vida de los pacientes afectados9. De acuerdo a Prusiner (premio nobel de medicina 1997), en todas las enfermedades neurodegenerativas hay algún tipo de proceso anormal de la función neuronal. Las enfermedades neurodegenerativas son la consecuencia de anormalidades en el proceso de ciertas proteínas que intervienen en el ciclo celular, por lo tanto da lugar al cúmulo de las mismas en las neuronas o en sus proximidades, disminuyendo o anulando sus funciones, como la enfermedad de Alzheimer y el mismo SXF. La proteína FMRP (Fragile Mental Retardation Protein), esencial para el desarrollo cognitivo normal, ha sido relacionada con la vía piramidal del dolor10-11-12. El Síndrome de X Frágil13-14 (SXF), se debe a la mutación del Gen (FMR-1). Como consecuencia de la mutación, el gen se inactiva y no puede realizar la función de sintetizar la proteína FMRP. Por su incidencia se le considera la primera causa de Deficiencia Mental Hereditaria sólo superada por el Síndrome de Down. La electroencefalografía (EEG) es el registro de la actividad bioeléctrica cerebral que ha traído el desarrollo diario de los estudios clínicos y experimentales para el descubrimiento, diagnóstico y tratamiento de un gran número de anormalidades neurológicas y fisiológicas del cerebro y el resto del sistema nervioso central (SNC) incluyendo el dolor. El objetivo de la presente investigación es por medio de un estudio multimodal, desarrollar nuevas formas de presentación diagnóstica mediante técnicas avanzadas de procesado de señal y de imagen, determinando así los vínculos entre las evaluaciones cognitivas y su correlación anatómica con la modulación al dolor presente en patologías relacionadas con proteína FMRP. Utilizando técnicas biomédicas (funcionalestructural) para su caracterización. Para llevar a cabo esta tarea hemos utilizado el modelo animal de ratón. Nuestros resultados en este estudio multimodal demuestran que hay alteraciones en las vías de dolor en el modelo animal FMR1-KO, en concreto en la modulación encefálica (dolor neuropático), los datos se basan en los resultados del estudio estructural (imagen histología), funcional (EEG) y en pruebas de comportamiento (Laberinto de Barnes). En la Histología se muestra una clara asimetría estructural en el modelo FMR1 KO con respecto al control WT, donde el hemisferio Izquierdo tiene mayor densidad de masa neuronal en KO hembras 56.7%-60.8%, machos 58.3%-61%, en WT hembras 62.7%-62.4%, machos 55%-56.2%, hemisferio derecho-izquierdo respectivamente, esto refleja una correlación entre hemisferios muy baja en los sujetos KO (~50%) con respecto a los control WT (~90%). Se encontró correlación significativa entre las pruebas de memoria a largo plazo con respecto a la asimetría hemisférica (r = -0.48, corregido <0,05). En el estudio de comportamiento también hay diferencias, los sujetos WT tuvieron 22% un de rendimiento en la memoria a largo plazo, mientras que en los machos hay deterioro de memoria de un 28% que se corresponden con la patología en humanos. En los resultados de EEG estudiados en el hemisferio izquierdo, en el área de la corteza insular, encuentran que la latencia de la respuesta al potencial evocado es menor (22vs32 15vs96seg), la intensidad de la señal es mayor para los sujetos experimentales FMR1 KO frente a los sujetos control, esto es muy significativo dados los resultados en la histología (140vs129 145vs142 mv). Este estudio multimodal corrobora que las manifestaciones clínicas del SXF son variables dependientes de la edad y el sexo. Hemos podido corroborar en el modelo animal que en la etapa de adulto, los varones con SXF comienzan a desarrollar problemas en el desempeño de tareas que requieren la puesta en marcha de la función ejecutiva central de la memoria de trabajo (almacenamiento temporal). En el análisis del comportamiento es difícil llegar a una conclusión objetiva, se necesitan más estudios en diferentes etapas de la vida corroborados con resultados histológicos. Los avances logrados en los últimos años en su estudio han sido muy positivos, de tal modo que se están abriendo nuevas vías de investigación en un conjunto de procesos que representan un gran desafío a problemas médicos, asistenciales, sociales y económicos a los que se enfrentan los principales países desarrollados, con un aumento masivo de las expectativas de vida y de calidad. Las herramientas utilizadas en el campo de las neurociencias nos ofrecen grandes posibilidades para el desarrollo de estrategias que permitan ser utilizadas en el área de la educación, investigación y desarrollo. La genética determina la estructura del cerebro y nuestra investigación comprueba que la ausencia de FMRP también podría estar implicada en la modulación del dolor como parte de su expresión patológica siendo el modelo animal un punto importante en la investigación científica fundamental para entender el desarrollo de anormalidades en el cerebro. ABSTRACT Pain is a common symptom in medical practice. In Spain, a study conducted in 2000 each medical professional treats an average of 181 patients with pain per month, most of them with chronic moderate pain. 7% -8% of the European population is affected and up to 5% can be serious, it is estimated to affect more than two million people in Spain. In Primary Care, patients with neuropathic pain have much higher rates of depression. Neuropathic pain is caused by damage or disease affecting the somatosensory system, is a public health problem with high labor costs, there are relatively unfamiliar with the peculiarities in diagnosis and treatment, failing that, the pain is perpetuated and becomes rebellious to treat, in most cases becomes chronic. The pathophysiological mechanisms are evolutionary, its a progressive, if untreated, causing severe impact on the quality of life of affected patients. According to Prusiner (Nobel Prize for Medicine 1997), all neurodegenerative diseases there is some abnormal process of neuronal function. Neurodegenerative diseases are the result of abnormalities in the process of certain proteins involved in the cell cycle, reducing or canceling its features such as Alzheimer's disease and FXS. FMRP (Fragile Mental Retardation Protein), is essential for normal cognitive development, and has been linked to the pyramidal tract pain. Fragile X Syndrome (FXS), is due to mutation of the gene (FMR-1). As a consequence of the mutation, the gene is inactivated and can not perform the function of FMRP synthesize. For its incidence is considered the leading cause of Mental Deficiency Hereditary second only to Down Syndrome. Electroencephalography (EEG) is the recording of bioelectrical brain activity, is a advancement of clinical and experimental studies for the detection, diagnosis and treatment of many neurological and physiological abnormalities of the brain and the central nervous system, including pain. The objective of this research is a multimodal study, is the development of new forms of presentation using advanced diagnostic techniques of signal processing and image, to determine the links between cognitive evaluations and anatomic correlation with pain modulation to this protein FMRP-related pathologies. To accomplish this task have used the mouse model. Our results in this study show alterations in multimodal pain pathways in FMR1-KO in brain modulation (neuropathic pain), the data are based on the results of the structural study (histology image), functional (EEG) testing and behavior (Barnes maze). Histology In structural asymmetry shown in FMR1 KO model versus WT control, the left hemisphere is greater density of neuronal mass (KO females 56.7% -60.8%, 58.3% -61% males, females 62.7% -62.4 WT %, males 55% -56.2%), respectively right-left hemisphere, this reflects a very low correlation between hemispheres in KO (~ 50%) subjects compared to WT (~ 90%) control. Significant correlation was found between tests of long-term memory with respect to hemispheric asymmetry (r = -0.48, corrected <0.05). In the memory test there are differences too, the WT subjects had 22% yield in long-term memory, in males there memory impairment 28% corresponding to the condition in humans. The results of EEG studied in the left hemisphere, in insular cortex area, we found that the latency of the response evoked potential is lower (22vs32 15vs96seg), the signal strength is higher for the experimental subjects versus FMR1 KO control subjects, this is very significant given the results on histology (140vs129 145vs142 mv). This multimodal study confirms that the clinical manifestations of FXS are dependent variables of age and sex. We have been able to corroborate in the animal model in the adult stage, males with FXS begin developing problems in the performance of tasks that require the implementation of the central executive function of working memory (temporary storage). In behavior analysis is difficult to reach an objective conclusion, more studies are needed in different life stages corroborated with histologic findings. Advances in recent years were very positive, being opened new lines of research that represent a great challenge to physicians, health care, social and economic problems facing the major developed countries, with a massive increase in life expectancy and quality. The tools used in the field of neuroscience offer us great opportunities for the development of strategies to be used in the area of education, research and development. Genetics determines the structure of the brain and our research found that the absence of FMRP might also be involved in the modulation of pain as part of their pathological expression being an important animal model in basic scientific research to understand the development of abnormalities in brain.

Overexpression of human copper,zinc-superoxide dismutase (SOD1) prevents postischemic injury

Superoxide and superoxide-derived oxidants have been hypothesized to be important mediators of postischemic injury. Whereas copper,zinc-superoxide dismutase, SOD1, efficiently dismutates superoxide, there has been controversy regarding whether increasing intracellular SOD1 expression would protect against or potentiate cellular injury. To determine whether increased SOD1 protects the heart from ischemia and reperfusion, studies were performed in a newly developed transgenic mouse model in which direct measurement of superoxide, contractile function, bioenergetics, and cell death could be performed. Transgenic mice with overexpression of human SOD1 were studied along with matched nontransgenic controls. Immunoblotting and immunohistology demonstrated that total SOD1 expression was increased 10-fold in hearts from transgenic mice compared with nontransgenic controls, with increased expression in both myocytes and endothelial cells. In nontransgenic hearts following 30 min of global ischemia a reperfusion-associated burst of superoxide generation was demonstrated by electron paramagnetic resonance spin trapping. However, in the transgenic hearts with overexpression of SOD1 the burst of superoxide generation was almost totally quenched, and this was accompanied by a 2-fold increase in the recovery of contractile function, a 2.2-fold decrease in infarct size, and a greatly improved recovery of high energy phosphates compared with that in nontransgenic controls. These results demonstrate that superoxide is an important mediator of postischemic injury and that increasing intracellular SOD1 dramatically protects the heart from this injury. Thus, increasing intracellular SOD1 expression may be a highly effective approach to decrease the cellular injury that occurs following reperfusion of ischemic tissues.