Resting and reactive blood pressure as predictors of ambulatory blood pressure in older hypertensive adults

Background. A review of the literature suggests that Hypertension (HTN) in older adults is associated with sympathetic stimulation that results in increasing blood pressure (BP) reactivity. If clinical assessment of BP captured sympathetic stimulation, it would be valuable for hypertension management. ^ Objectives. The study examined whether reactive change scores from a short BPR protocol, resting blood pressure (BP), or resting pulse pressure (PP) is a better predictor of 24 hour ambulatory BP and BP load in cardiac patients. ^ Method. The study used a single-group design, with both an experimental clinical component and an observational field component. Both components used repeated measurement methods. The study population consisted of 45 adult patients with a mean age of 64.6 ± 8.5 years who were diagnosed with cardiac disease and who were taking anti-hypertensive medication. Blood pressure reactivity was operationalized with a speech protocol. During the speech protocol, BP was measured with an automatic device (Dinamap 825XT) while subjects talked about their health and about their usual day. Twenty-four hour ambulatory BP measurement (Spacelabs 90207 monitor) followed the speech protocol. ^ Results. Resting SBP and resting PP were significant predictors of 24-hour SBP, and resting SBP was a significant predictor of SBP load. No predictors were significant of 24-hour DBP or DBP load. ^ Conclusions. Initial resting BP and PP may be used in clinical settings to assess hypertension management. Future studies are necessary to confirm the ability of resting BP to predict ABP and BP load in older, medicated hypertensives. ^

Effects of Aging and Moderate Calorie Restriction on the Reproductive Axis of the Male Rhesus Macaque (Macaca mulatta)

Calorie restriction (CR) has been established as the only non-genetic method of altering longevity and attenuating biological changes associated with aging. This nutritional paradigm has been effective in nematodes, flies, rodents, dogs and possibly non-human primates. Its long history notwithstanding, little is known regarding the exact mechanism(s) of CR action or its potential impact on the hypothalamic-pituitary-gonadal (HPG) axis. The objectives of this project were to: 1) analyze neuroendocrine changes to the HPG axis that occur with aging and 2) evaluate the effects of moderate CR on reproductive function in male rhesus macaques. Pituitary gene expression profiling, semi-quantitative RT-PCR (sqRT-PCR) and immunohistochemistry showed circadian clock mechanism components present in three age categories of macaques, demonstrated age differences in expression for Per2, indicated differential expression of Per2 and Bmal1 at opposing time points and revealed daily rhythmic expression of REV-ERBα protein. These data indicate the ability of the macaque pituitary to express core-clock genes, their protein products, and to do so in a 24-hour rhythm. Young Adult CON and CR pituitary gene expression profiles detected potential differential expression in <150 probesets. A decline in>TSHR and CGA was detected in CR macaques as measured by sqRT-PCR. Other genes investigated showed no diet-induced changes. Young Adult CON and CR testicular gene expression profiles detected potential differential expression in <300 probesets although mRNA expression was not altered based on sqRT-PCR and real-time RT-PCR. Age-related>and/or diet-induced changes in HSD17β3, INSL3, CSNK1E and CGA were observed in a separate experiment with CGA in Old Adult CR subjects returning to youthful levels. Semen samples were collected from Young Adult CON and CR macaques. Normal spermiogram measures, ZP-binding, AR assay and SCSA^® were conducted and indicated no differences between CON and CR-treated animals. Both groups exhibited similar daily testosterone profiles with no differences in mean or maximum levels; however, daily minimum testosterone levels were lower in CON animals. It appears that moderate CR had limited impact on neuroendocrine or reproductive function in male rhesus macaques based on our selected endpoints. Thus, advantageous CR health benefits can be achieved without obvious negative consequences to the HPG axis.

Type I hypersensitivity and its pharmacological modulation in {\it Trichinella\/}-induced intestinal fluid secretion and rapid rejection of the parasite in immunized rats

Studies were performed to test the hypothesis that type I hypersensitivity underlies worm induced intestinal fluid secretion and the rapid rejection of Trichinella spiralis from immunized rats, and the two events may be related in a cause-effect manner.^ Two approaches were taken. One was to determine whether inhibition of anaphylaxis-mediated Cl$\sp{-}$ and fluid secretion accompanying a secondary infection impedes worm rejection from immune hosts. The other was to determine whether induction of intestinal fluid secretion in nonimmune hosts interfered with worm establishment. In both studies, fluid secretion was measured volumetrically 30 min after a challenge infection and worms were counted.^ In immunized rats indomethacin did not affect the worm-induced fluid secretion when used alone, despite inhibiting mucosal prostaglandin synthesis. Fluid secretion was reduced by treatment with diphenhydramine and further reduced by the combination of diphenhydramine and indomethacin. The paradoxical effects of indomethacin when used alone compared with its coadministration with diphenhydramine is explained by the enhancing effect of indomethacin on histamine release. Abolishing net fluid secretion in these studies had no effect on rapid worm rejection in immune hosts.^ Worm establishment was reduced in recipients of immune serum containing IgE antibodies. Net intestinal fluid secretion induced in normal rats by PGE$\sb2$, cholera toxin, or hypertonic mannitol solution had no effect on worm establishment compared with untreated controls.^ In a related experiment, worm-induced intestinal fluid secretion and worm rejection in immune rats were partially blocked by concurrent injection with 5-HT$\sb2$ and 5-HT$\sb3$ blockers (Ketanserin and MDL-72222), suggesting that 5-HT is involved. This possible involvement was supported in that treatment of nonimmune rats with 5-HT significantly inhibited worm establishment in the intestine.^ Results indicate that anaphylaxis is the basis for both worm-induced intestinal fluid secretion and rapid rejection of T. spiralis in immune rats, but these events are independent of one another. 5-HT is a possible mediator of worm rejection, however, its mechanism of action is related to something other than fluid secretion. ^

Suppression by {\it Trypanosoma brucei\/} of anaphylaxis-mediated ion transport in the small intestine of rats

Most tissue-invasive parasitic helminths prime for type 1 hypersensitivity or anaphylaxis during some phase of their life cycles. A prototype in this regard is the nematode Trichinella spiralis. Blood protozoa capable of tissue invasion, such as Trypanosoma brucei, might also be expected to prime for the expression of anaphylaxis. However, this response is usually absent in protozoal infections. The hypothesis tested was that failure of hosts infected with T.brucei to express anaphylaxis is related to this parasite's ability to selectively down-regulate immunoglobulin E (IgE) production, and not to an innate lack of allergenicity on the part of T.brucei-derived antigens. This hypothesis was tested by studying in the intestine of rats, antigen-induced Cl$\sp-$ secretion, which results from a local anaphylactic response mediated by IgE and mucosal mast cells. The Cl$\sp-$ secretory response can be primed either by infection with T.spiralis or by the parenteral administration of antigen. Anaphylaxis-induced Cl$\sp-$ secretion is expressed in vitro, and can be quantified electrophysiologically, as a change in transmural short-circuit current when sensitized intestine is mounted in Ussing chambers and challenged with the sensitizing antigen.^ Rats injected parenterally with trypanosome antigen elicited intestinal anaphylaxis in response to antigenic challenge. In contrast, the intestine of rats infected with T.brucei failed to respond to challenge with trypanosome antigen. Infection with T.brucei also suppressed antigen-induced Cl$\sp-$ secretion in rats sensitized and challenged with various antigens, including T.spiralis antigen. However, T.brucei infection did not inhibit the anaphylactic response in rats concomitantly infected with T.spiralis. Relative to the anaphylactic mediators, T.brucei infection blocked production of IgE in rats parenterally injected with antigen but not in T.spiralis-infected hosts. Also, the mucosal mastocytosis normally associated with trichinosis was unaffected by the trypanosome infection. These results support the conclusion that the failure to express anaphylaxis-mediated Cl$\sp-$ secretion in T.brucei infected rats, is due to this protozoan's ability to inhibit IgE production and not to the lack of allergenicity of trypanosome antigens. ^

Early stages of experimental colon carcinogenesis inhibit mast cell-dependent mucosal immune function of the distal colon

Inhibition of local host immune reactions is one mechanism contributing to tumor progression. To determine if alterations in local immune functioning occur during colon carcinogenesis, a model mucosal immune response, type I hypersensitivity against the intestinal parasite Trichinella spiralis, was first characterized in normal mice and then examined during experimental colon carcinogenesis. Segments of sensitized colon mounted in Ussing chambers and challenged with T. spiralis-derived antigen resulted in a rise in short-circuit current ($\rm\Delta I\sb{sc}$) that was antigen-specific and inhibited by furosemide, implicating epithelial Cl$\sp-$ secretion as the ionic mechanism. The immune-regulated Cl$\sp-$ secretion by colonic epithelial cells required the presence of mast cells with surface IgE. Inhibition of potential anaphylactic mediators with various pharmacological agents in vitro implicated prostaglandins and leukotrienes as the principal mediators of the antigen-induced $\rm\Delta I\sb{sc}$, with 5-hydroxytryptamine also playing a role. Distal colon from immune mice fed an aspirin-containing diet (800 mg/kg powdered diet) ad libitum for 6 wk had a decreased response to antigen, confirming the major role of prostaglandins in generating the colonic I$\sb{\rm sc}$. To determine the effects of early stages of colon carcinogenesis on this mucosal immune response, mice were immunized with T. spiralis 1 day after or 8 wk prior to the first of 6 weekly injections of the procarcinogen 1,2-dimethylhydrazine (DMH). Responsiveness to antigenic challenge was suppressed in the distal colon 4-6 wk after the final injection of DMH. One injection of DMH was not sufficient to inhibit antigen responsiveness. The colonic epithelium remained sensitive to direct stimulation by exogenous Cl$\sp-$ secretagogues. Decreased antigen-induced $\rm\Delta I\sb{sc}$ in the distal colon was not due to systemic immune suppression by DMH, as the proximal colon and jejunum maintained responsiveness to antigen. Also, rejection of a secondary T. spiralis infection from the small intestine was not altered. Tumors eventually developed 25-30 wk after the final injection of DMH only in the distal portions of the colon. These results suggest that early stages of DMH-induced colon carcinogenesis manipulate the microenvironment such that mucosal immune function, as measured by immune-regulated Cl$\sp-$ secretion, is suppressed in the distal colon, but not in other regions of the gut. Future elucidation of the mechanisms by which this localized inhibition of immune-mediated ion transport occurs may provide possible clues to the microenvironmental changes necessary for tumor progression in the distal colon. ^

Regulation and function of {\it Xparaxis\/} during the development of paraxial mesoderm in {\it Xenopus laevis\/}

Genes of the basic helix-loop-helix transcription factor family have been implicated in many different developmental processes from neurogenesis to myogenesis. The recently cloned bHLH transcription factor, paraxis, has been found to be expressed in the paraxial mesoderm of the mouse suggesting a role for paraxis in the development of this mesodermal subtype which gives rise to the axial muscle, skeleton, and dermis of the embryo. In order to perform in vivo gain of function assays and obtain a better understanding of the possible roles of paraxis in mesodermal and somitic development, we have successfully identified homologues of paraxis in the frog, Xenopus laevis, where the process of mesodermal induction and development is best understood. The two homologues, Xparaxis-a and Xparaxis-b, are conserved with respect to their murine homologue in structure and expression within the embryo. Xparaxis genes are expressed immediately after gastrulation in the paraxial mesoderm of Xenopus embryos and are down regulated in the myotome of the mature somite with continued expression in the undifferentiated dermatome. Overexpression of Xparaxis-b in Xenopus embryos caused defects in the organization and morphology of the somites. This effect was not dependent on DNA binding of Xparaxis but is likely due to its dimerization with other bHLH factors. Co-injections with XE12 did not diminish the effects indicating that the defects were not the result of limiting amounts of XE12. We also demonstrated that Xparaxis does not cause obvious defects in the cell adhesions and movements required for proper mesoderm patterning during gastrulation. The paraxis proteins also lacked the ability to activate transcription as GAL4 fusion proteins in a GAL4 reporter assay, indicating that the genes may function more as modulators of the activity of dimerization partners than as positively acting cell determination factors. In agreement with this, Xparaxis is regulated in response to other pathways of bHLH gene action, in that XE12 can activate Xparaxis-b, in vivo. In addition we show regulation of Xparaxis in response to mMyoD induced myogenesis pathways, again suggesting Xparaxis plays an important role in the patterning and organization of the paraxial mesoderm. ^

A study of the relationship between physical activity and cardiovascular fitness and coronary heart disease risk factors in female adolescents

The association of measures of physical activity with coronary heart disease (CHD) risk factors in children, especially those for atherosclerosis, is unknown. The purpose of this study was to determine the association of physical activity and cardiovascular fitness with blood lipids and lipoproteins in pre-adolescent and adolescent girls.^ The study population was comprised of 131 girls aged 9 to 16 years who participated in the Children's Nutrition Research Center's Adolescent Study. The dependent variables, blood lipids and lipoproteins, were measured by standard techniques. The independent variables were physical activity measured as the difference between total energy expenditure (TEE) and basal metabolic rate (BMR), and cardiovascular fitness, VO$\rm\sb{2max}$(ml/min/kg). TEE was measured by the doubly-labeled water (DLW) method, and BMR by whole-room calorimetry. Cardiovascular fitness, VO$\rm\sb{2max}$(ml/min/kg), was measured on a motorized treadmill. The potential confounding variables were sexual maturation (Tanner breast stage), ethnic group, body fat percent, and dietary variables. A systematic strategy for data analysis was used to isolate the effects of physical activity and cardiovascular fitness on blood lipids, beginning with assessment of confounding and interaction. Next, from regression models predicting each blood lipid and controlling for covariables, hypotheses were evaluated by the direction and value of the coefficients for physical activity and cardiovascular fitness.^ The main result was that cardiovascular fitness appeared to be more strongly associated with blood lipids than physical activity. An interaction between cardiovascular fitness and sexual maturation indicated that the effect of cardiovascular fitness on most blood lipids was dependent on the stage of sexual maturation.^ A difference of 760 kcal/d physical activity (which represents the difference between the 25th and 75th percentile of physical activity) was associated with negligible differences in blood lipids. In contrast, a difference in 10 ml/min/kg of VO$\rm\sb{2max}$ or cardiovascular fitness (which represents the difference between the 25th and 75th percentile in cardiovascular fitness) in the early stages of sexual maturation was associated with an average positive difference of 15 mg/100 ml ApoA-1 and 10 mg/100 ml HDL-C. ^

Genes to blood pressure: The effects of variation in genes of the renin-angiotensin system on the hormonal and renal control of blood pressure

Objective. Essential hypertension affects 25% of the US adult population and is a leading contributor to morbidity and mortality. Because BP is a multifactorial phenotype that resists simple genetic analysis, intermediate phenotypes within the complex network of BP regulatory systems may be more accessible to genetic dissection. The Renin-Angiotensin System (RAS) is known to influence intermediate and long-term blood pressure regulation through alterations in vascular tone and renal sodium and fluid resorption. This dissertation examines associations between renin (REN), angiotensinogen (AGT), angiotensin-converting enzyme (ACE) and angiotensin II type 1 receptor (AT1) gene variation and interindividual differences in plasma hormone levels, renal hemodynamics, and BP homeostasis.^ Methods. A total of 150 unrelated men and 150 unrelated women, between 20.0 and 49.9 years of age and free of acute or chronic illness except for a history of hypertension (11 men and 7 women, all off medications), were studied after one week on a controlled sodium diet. RAS plasma hormone levels, renal hemodynamics and BP were determined prior to and during angiotensin II (Ang II) infusion. Individuals were genotyped by PCR for a variable number tandem repeat (VNTR) polymorphism in REN, and for the following restriction fragment length polymorphisms (RFLP): AGT M235T, ACE I/D, and AT1 A1166C. Associations between clinical measurements and allelic variation were examined using multiple linear regression statistical models.^ Results. Women homozygous for the AT1 1166C allele demonstrated higher intracellular levels of sodium (p = 0.044). Men homozygous for the AGT T235 allele demonstrated a blunted decrement in renal plasma flow in response to Ang II infusion (p = 0.0002). There were no significant associations between RAS gene variation and interindividual variation in RAS plasma hormone levels or BP.^ Conclusions. Rather than identifying new BP controlling genes or alleles, the study paradigm employed in this thesis (i.e., measured genes, controlled environments and interventions) may provide mechanistic insight into how candidate genes affect BP homeostasis. ^

Body fat distribution and serum lipids in children and adolescents

Previous studies of normal children have linked body fat but not body fat distribution (BFD), to higher blood pressures, lipids, and insulin resistance (Berenson et al., 1988) BFD is a well-established risk factor for cardiovascular disease in adults (Björntorp, 1988). This study investigates the relation of BFD and serum lipids at baseline in children from Project HeartBeat!, a study of the growth and development of cardiovascular risk factors in 678 children in three cohorts measured initially at ages 8, 11, and 14 years. Initially, two of four indices of BFD were significantly related to the lipids: ratio of upper to lower body skinfolds (ln US:LS) and conicity (C Index). A factor analysis reduced the information in the serum lipids to two vectors: (1) total cholesterol + LDL-cholesterol and (2) HDL-cholesterol − triglycerides, which together accounted for 85% of the lipid variation. Using each serum lipid and vector as separate dependent variables, linear and quadratic regression models were constructed to examine the predictive ability of the two BFD variables, controlling for total body fat, gender, ethnicity (Black, non-Black) and maturation. Linear models provided an acceptable fit. Percent body fat (%BF) was a significant predictor in each and every lipid model, independent of age, maturation, or ethnicity (p ≤ 0.05). No BFD variable entered the equation for total or LDL-cholesterol, although there was a significant maturity by BFD interaction for LDL (ln US:LS was a significant predictor in more mature individuals). Both %BF and BFD (by way of Conicity) were significant predictors of HDL-cholesterol and triglycerides (p ≤ 0.01). All models were statistically significant at a high level (p ≤ 0.01), but adjusted R 2's for all models were low (0.05–0.15). Body fat distribution is a significant predictor of lipids in normal children, but secondarily to %BF, and for LDL-cholesterol in particular, the relation is dependent on maturity status. ^

Alteration of CEACAM1 gene transcription in prostate cancer cells: The role of AR, Sp2, and HDAC

Cell to cell adhesion molecule (CEACAM1), a type II tumor suppressor, has been found to be down-regulated in prostate cancer cells. The mechanism that causes CEACAM1's down-regulation in tumorigenesis is unknown. Here we show that the transcriptional activity of CEACAM1 is decreased in prostate cancer cells. This decrease is not due to methylation of the CEACAM1's promoter, but rather to the alteration of transcription factors regulating CEACAM1 expression. ^ Since androgen/androgen receptors (AR) are potent regulators of prostate growth and differentiation, their role on CEACAM1 gene transcription was examined. The androgen receptor could directly increase CEACAM1 transcriptional activity in a ligand dependent manner by interacting with an AR consensus element that resides in the CEACAM1 promoter. However, AR binding to the CEACAM1 promoter is not related to the loss of CEACAM1 during prostate cancer progression. ^ Further analysis enabled us to determine the particular region in the CEACAM1 promoter that mediates a decrease in CEACAM1 transcriptional activity in prostate cancer cells. Upon further examination, we found that this CEACAM1 promoter region interacts with the Sp1, Sp2, and Sp3 transcription factors. However, only Sp2 expression was found to increase in prostate cancer cells. Inhibiting Sp2 from binding to the CEACAM1 promoter caused an increase in CEACAM1 transcriptional activity in prostate cancer cells. In addition, over-expressing Sp2 in normal prostate cells resulted in a decrease in CEACAM1 transcriptional activity and endogenous protein expression. These observations suggest that Sp2 is a transcription repressor of CEACAM1. Furthermore, prostate cancer cells treated with trichostatin A (TSA), a specific histone deacetylase (HDAC) inhibitor, activated CEACAM1 transcriptional activity. This implies that HDACs are involved in CEACAM1 transcriptional activity. Mutation of the Sp2 DNA binding region on the CEACAM1 promoter inhibited TSA activation of CEACAM1 transcriptional activity. This indicates that HDACs inhibit CEACAM1 transcriptional activity through Sp2. Base on these results, we propose that Sp2 is critical for down-regulating CEACAM1 expression, and one mechanism by which Sp2 represses CEACAM1 expression is by recruiting HDAC to the CEACAM1 promoter in prostate cancer cells. Collectively, these findings provide novel insights into mechanisms that cause the down-regulation of CEACAM1 expression in prostate cancer cells. ^

17-beta estradiol and progesterone-induced alterations in human type-1/type-2 cytokine balance and the role of costimulatory and apoptotic mechanisms

Evidence suggests that sex-based differences in immune function may predispose women to numerous hypersensitivity conditions such as Systemic lupus erythematosus (SLE), Hashimoto's thyroiditis and asthma. To date, the exact mechanisms of sexual dimorphism in immunity are not fully characterized but sex hormones such as 17-β estradiol (E2) and progesterone (PR) are believed to be involved. Since E2 and PR may modulate the production of critical regulatory cytokines, we sought to characterize their effects on the in vitro human type-1/type-2 cytokine balance. We hypothesized that E2 and/or PR vary cytokine production and influence costimulatory molecule expression and apoptosis. We first described the effect of E2 and/or PR on type-1 (IFN-γ and IL-12) and type-2 (IL-4 and IL-10) cytokine production by human peripheral blood mononuclear cells (PBMC) treated with various T-lymphocyte and monocyte stimuli. E2 and/or PR were each used at concentrations similar to those found at the maternal-fetal interface during pregnancy. At this dose, E2 increased IFN-γ and IL-12 production and PR decreased IFN-γ production and tended to increase IL-4 production. Furthermore, the combination of E2+PR decreased IL-12 production. This suggests that E2 shifts the type-1/type-2 cytokine balance towards a type-1 response and that PR and E2+PR shift the balance towards a type-2 response. Next, we used intracellular cytokine detection to demonstrate that E2 and/or PR are capable of altering cytokine production of CD3+ T-cells and the CD3+CD4+ and CD3+CD8+ subsets. In addition, we used the H9 T-lymphocyte cell line and the THP-1 monocyte cell line to show that E2 and/or PR can induce cytokine effects in both T-cells and monocytes independent of their interaction. Lastly, we determined the effect of E2 and/or PR on costimulatory molecule expression and apoptosis as potential mechanisms for the cytokine-induced alterations. E2 increased and PR decreased CD80 expression on THP-1 cells and PR and E2+PR decreased CD28 expression in PBMC and Jurkat cells. Furthermore, E2, PR and E2+PR increased Fas-mediated apoptosis in Jurkat cells and E2 increased FasL expression on THP-1 cells. Thus, E2 and/or PR may alter the cytokine balance by modulating the CD28/CD80 costimulatory pathway and apoptosis. ^

Role of organic cation transporters in the renal secretion of nucleoside analogs

The mammalian kidney maintains homeostasis of the extracellular environment and eliminates toxic substances from the body, in part via secretion by the organic cation transporters (OCT). Some nucleosides are also secreted by the kidney. Previous work indicated that the deoxyadenosine analog, 2′ -deoxytubercidin (dTub), is secreted by mouse kidney through the OCTs. This study examines the role of OCTs in the renal secretion of dTub and other nucleoside analogs. ^ Using the Xenopus laevis oocyte expression system, the basolateral type rat organic cation transporter rOCT1 was shown to transport dTub and other nucleosides. The positive charged form of dTub (dTub +) appears to be the substrate for rOCT1. Tetraethylammonium (TEA) and dTub competitively inhibit the other's uptake by rOCT1 in a manner consistent with their interaction at a common site. Although 67% homologous with rOCT1, rOCT2 does not mediate the uptake of these nucleosides. Kinetic studies demonstrated the difference in substrate specificity between rOCT1 and rOCT2 to be largely due to a poor affinity of rOCT2 for dTub+. This difference in affinity is located within transmembrane domains 2–7 as determined by chimeric constructs. ^ OCT1 knockout mice were used to evaluate the role of OCT1 in the renal secretion of dTub. No significant difference in tissue distribution and urinary excretion of dTub was observed between the knockout and wild-type mice, indicating that OCT1 is not necessary for the renal secretion of dTub. Apical transporters are postulated to participate in its active secretion. To characterize a possible apical transporter, we screened several renal cell lines for a nucleoside-sensitive OCT. American opossum kidney proximal tubule cells (OK) express a TEA efflux transporter that is inhibited by dTub and other nucleoside analogs. This carrier is metabolic-dependent and distinct from the cloned OCTs to date, i.e. it is sodium- and proton-independent. In conclusion, dTub is a good substrate for OCT1; however, this OCT is not necessary for its renal secretion in mice. The novel TEA efflux transporter identified in OK cells is likely to participate in the renal secretion of dTub and perhaps other nucleoside analogs. ^

Vasculogenesis: A process of vessel formation and its role during Ewing's sarcoma development

Vasculogenesis is the process by which Endothelial Precursor Cells (EPCs) form a vasculature. This process has been traditionally regarded as an embryological process of vessel formation. However, as early as in the 60's the concept of postnatal vasculogenesis was introduced, with a strong resurface of this idea in recent years. Similarly, previous work on a mouse skin tumor model provided us with the grounds to consider the role of vasculogenesis during tumor formation. ^ We examined the contribution of donor bone marrow (BM)-derived cells to neovascularization in recipient nude mice with Ewing's sarcoma. Ewing's sarcoma is a primitive neuroectodermal tumor that most often affects children and young adults between 5 and 30 years of age. Despite multiple attempts to improve the efficacy of chemotherapy for the disease, the 2-year metastases-free survival rate for patients with Ewing's sarcoma has not improved over the past 15 years. New therapeutic approaches are therefore needed to reduce the mortality rate. ^ The contribution of BM endothelial precursor cells in the development of Ewing's sarcoma was examined using different strategies to track the donor-derived cells. Using a BMT model that takes advantage of MHC differences between donor and recipient mice, we have found that donor BM cells were involved in the formation of Ewing's sarcoma vasculature. ^ Cells responsible for this vasculogenesis activity may be located within the stem cell population of the murine BM. These stem cells would not only generate the hematopoietic lineage but they would also generate ECs. Bone marrow SP (Side Population) cells pertain to a subpopulation that can be identified using flow cytometric analysis of Hoechst 33342-stained BM. This population of cells has HSC activity. We have tested the ability of BM SP cells to contribute to vasculogenesis in Ewing's sarcoma using our MHC mismatched transplant model. Mice transplanted with SP cells developed tumor neovessels that were derived from the donor SP cells. Thus, SP cells not only replenished the hematopoietic system of the lethally irradiated mice, but also differentiated into a non-hematopoietic cell lineage and contributed to the formation of the tumor vasculature. ^ In summary, we have demonstrated that BM-derived cells are involved in the generation of the new vasculature during the growth of Ewing's sarcoma. The finding that vasculogenesis plays a role in Ewing's sarcoma development opens the possibility of using genetically modified BM-derived cells for the treatment of Ewing's sarcomas. ^

Molecular and genetic analyses of Fgf signaling during cardiac outflow tract development

Epithelial-mesenchymal tissue interactions regulate the development of derivatives of the caudal pharyngeal arches (PAs) to govern the ultimate morphogenesis of the aortic arch and outflow tract (OFT) of the heart. Disruption of these signaling pathways is thought to contribute to the pathology of a significant proportion of congenital cardiovascular defects in humans. In this study, I tested whether Fibroblast Growth Factor 15 (Fgf15), a secreted signaling molecule expressed within the PAs, is an extracellular mediator of tissue interactions during PA and OFT development. Analyses of Fgf15−/− mouse embryonic hearts revealed abnormalities primarily localized to the OFT, correlating with aberrant cardiac neural crest cell behavior. The T-box-containing transcription factor Tbx1 has been implicated in the cardiovascular defects associated with the human 22q11 Deletion Syndromes, and regulates the expression of other Fgf family members within the mouse PAs. However, expression and genetic interaction studies incorporating mice deficient for Tbx1, its upstream regulator, Sonic Hedgehog (Shh), or its putative downstream effector, Fgf8, indicated that Fgf15 functions during OFT development in a manner independent of these factors. Rather, analyses of compound mutant mice indicated that Fgf15 and Fgf9, an additional Fgf family member expressed within the PAs, genetically interact, providing insight into the factors acting in conjunction with Fgf15 during OFT development. Finally, in an effort to further characterize this Fgf15-mediated developmental pathway, promoter deletion analyses were employed to isolate a 415bp sequence 7.1Kb 5′ to the Fgf15 transcription start site both necessary and sufficient to drive reporter gene expression within the epithelium of the PAs. Sequence comparisons among multiple mammalian species facilitated the identification of evolutionarily conserved potential trans-acting factor binding sites within this fragment. Subsequent studies will investigate the molecular pathway(s) through which Fgf15 functions via identification of factors that bind to this element to govern Fgf15 gene expression. Furthermore, targeted deletion of this element will establish the developmental requirement for pharyngeal epithelium-derived Fgf15 signaling function. Taken as a whole, these data demonstrate that Fgf15 is a component of a novel, Tbx1-independent molecular pathway, functioning within the PAs in a manner cooperative with Fgf9, required for proper development of the cardiac OFT. ^

The genetic contribution of the major histocompatibility complex to bleomycin-induced lung injury

Pulmonary fibrosis (PF) is the result of a variety of environmental and cancer treatment related insults and is characterized by excessive deposition of collagen. Gas exchange in the alveoli is impaired as the normal lung becomes dense and collapsed leading to a loss of lung volume. It is now accepted that lung injury and fibrosis are in part genetically regulated. ^ Bleomycin is a chemotherapeutic agent used for testicular cancer and lymphomas that induces significant pulmonary toxicity. We delivered bleomycin to mice subcutaneously via a miniosmotic pump in order to elicit lung injury (LI) and quantified the %LI morphometrically using video imaging software. We previously identified a quantitative trait loci, Blmpf-1(LOD=17.4), in the Major Histocompatibility Complex (MHC), but the exact genetic components involved have remained unknown. ^ In the current studies, Blmpf-1 was narrowed to an interval spanning 31.9-32.9Mb on Chromosome 17 using MHC Congenic mice. This region includes the MHC Class II and III genes, and is flanked by the TNF-alpha super locus and MHC Class I genes. Knockout mice of MHC Class I genes (B2mko), MHC Class II genes (Cl2ko), and TNF-alpha (TNF-/-) and its receptors (p55-/-, p75-/-, and p55/p75-/-) were treated with bleomycin in order to ascertain the role of these genes in the pathogenesis of lung injury. ^ Cl2ko mice had significantly better survival and %LI when compared to treated background BL/6 (B6, P<.05). In contrast, B2mko showed no differences in survival or %LI compared to B6. This suggests that the MHC Class II locus contains susceptibility genes for bleomycin-induced lung injury. ^ TNF-alpha, a Class III gene, was examined and it was found that TNF-/- and p55-/- mice had higher %LI and lower survival when compared to B6 (P<.05). In contrast, p75-/- mice had significantly reduced %LI when compared to TNF-/-, p55-/-, and B6 mice as well as higher survival (P<.01). These data contradict the current paradigm that TNF-alpha is a profibrotic mediator of lung injury and suggest a novel and distinct role for the p55 and p75 receptors in mediating lung injury. ^