A Villin-Driven Fxr Transgene Modulates Enterohepatic Bile Acid Homeostasis and Response to an n-6-Enriched High-Fat Diet.

A diet high in n-6 polyunsaturated fatty acids (PUFAs) may contribute to inflammation and tissue damage associated with obesity and pathologies of the colon and liver. One contributing factor may be dysregulation by n-6 fatty acids of enterohepatic bile acid (BA) metabolism. The farnesoid X receptor (FXR) is a nuclear receptor that regulates BA homeostasis in the liver and intestine. This study aims to compare the effects on FXR regulation and BA metabolism of a palm oil-based diet providing 28% energy (28%E) from fat and low n-6 linoleic acid (LA, 2.5%E) (CNTL) with those of a soybean oil-based diet providing 50%E from fat and high (28%E) in LA (n-6HFD). Wild-type (WT) littermates and a transgenic mouse line overexpressing the Fxrα1 isoform under the control of the intestine-specific Villin promoter (Fxrα1TG) were fed the CNTL or n-6HFD starting at weaning through 16 weeks of age. Compared to the CNTL diet, the n-6HFD supports higher weight gain in both WT and FxrαTG littermates; increases the expression of Fxrα1/2, and peroxisome proliferator-activated receptor-γ1 (Pparγ1) in the small intestine, Fxrα1/2 in the colon, and cytochrome P4507A1 (Cyp7a1) and small heterodimer protein (Shp) in the liver; and augments the levels of total BA in the liver, and primary chenodeoxycholic (CDCA), cholic (CA), and ß-muricholic (ßMCA) acid in the cecum. Intestinal overexpression of the Fxra1TG augments expression of Shp and ileal bile acid-binding protein (Ibabp) in the small intestine and Ibabp in the proximal colon. Conversely, it antagonizes n-6HFD-dependent accumulation of intestinal and hepatic CDCA and CA; hepatic levels of Cyp7a1; and expression of Pparγ in the small intestine. We conclude that intestinal Fxrα1 overexpression represses hepatic de novo BA synthesis and protects against n-6HFD-induced accumulation of human-specific primary bile acids in the cecum.

Inactivation of Adenomatous Polyposis Coli Reduces Bile Acid/Farnesoid X Receptor Expression through Fxr gene CpG Methylation in Mouse Colon Tumors and Human Colon Cancer Cells.

BACKGROUND: The farnesoid X receptor (FXR) regulates bile acid (BA) metabolism and possesses tumor suppressor functions. FXR expression is reduced in colorectal tumors of subjects carrying inactivated adenomatous polyposis coli (APC). Identifying the mechanisms responsible for this reduction may offer new molecular targets for colon cancer prevention. OBJECTIVE: We investigated how APC inactivation influences the regulation of FXR expression in colonic mucosal cells. We hypothesized that APC inactivation would epigenetically repress nuclear receptor subfamily 1, group H, member 4 (FXR gene name) expression through increased CpG methylation. METHODS: Normal proximal colonic mucosa and normal-appearing adjacent colonic mucosa and colon tumors were collected from wild-type C57BL/6J and Apc-deficient (Apc(Min) (/+)) male mice, respectively. The expression of Fxr, ileal bile acid-binding protein (Ibabp), small heterodimer partner (Shp), and cyclooxygenase-2 (Cox-2) were determined by real-time polymerase chain reaction. In both normal and adjacent colonic mucosa and colon tumors, we measured CpG methylation of Fxr in bisulfonated genomic DNA. In vitro, we measured the impact of APC inactivation and deoxycholic acid (DCA) treatment on FXR expression in human colon cancer HCT-116 cells transfected with silencing RNA for APC and HT-29 cells carrying inactivated APC. RESULTS: In Apc(Min) (/+) mice, constitutive CpG methylation of the Fxrα3/4 promoter was linked to reduced (60-90%) baseline Fxr, Ibabp, and Shp and increased Cox-2 expression in apparently normal adjacent mucosa and colon tumors. Apc knockdown in HCT-116 cells increased cellular myelocytomatosis (c-MYC) and lowered (∼50%) FXR expression, which was further reduced (∼80%) by DCA. In human HCT-116 but not HT-29 colon cancer cells, DCA induced FXR expression and lowered CpG methylation of FXR. CONCLUSIONS: We conclude that the loss of APC function favors the silencing of FXR expression through CpG hypermethylation in mouse colonic mucosa and human colon cells, leading to reduced expression of downstream targets (SHP, IBABP) involved in BA homeostasis while increasing the expression of factors (COX-2, c-MYC) that contribute to inflammation and colon cancer.

2-Photon Characterization of Optical Proteolytic Beacons for Imaging Changes in Matrix-Metalloprotease Activity in a Mouse Model of Aneurysm.

Abdominal aortic aneurysm is a multifactorial disease that is a leading cause of death in developed countries. Matrix-metalloproteases (MMPs) are part of the disease process, however, assessing their role in disease initiation and progression has been difficult and animal models have become essential. Combining Förster resonance energy transfer (FRET) proteolytic beacons activated in the presence of MMPs with 2-photon microscopy allows for a novel method of evaluating MMP activity within the extracellular matrix (ECM). Single and 2-photon spectra for proteolytic beacons were determined in vitro. Ex vivo experiments using the apolipoprotein E knockout angiotensin II-infused mouse model of aneurysm imaged ECM architecture simultaneously with the MMP-activated FRET beacons. 2-photon spectra of the two-color proteolytic beacons showed peaks for the individual fluorophores that enable imaging of MMP activity through proteolytic cleavage. Ex vivo imaging of the beacons within the ECM revealed both microstructure and MMP activity. 2-photon imaging of the beacons in aneurysmal tissue showed an increase in proteolytic cleavage within the ECM (p<0.001), thus indicating an increase in MMP activity. Our data suggest that FRET-based proteolytic beacons show promise in assessing MMP activity within the ECM and will therefore allow future studies to identify the heterogeneous distribution of simultaneous ECM remodeling and protease activity in aneurysmal disease.

Acute Elution of TGFß2 Affects the Smooth Muscle Cells in a Compliance-Matched Vascular Graft.

Transforming growth factor beta 2 (TGFß2) is a pleiotropic growth factor that plays a vital role in smooth muscle cell (SMC) function. Our prior in vitro work has shown that SMC response can be modulated with TGFß2 stimulation in a dose dependent manner. In particular, we have shown that increasing concentrations of TGFß2 shift SMCs from a migratory to a synthetic behavior. In this work, electrospun compliance-matched and hypocompliant TGFß2-eluting tissue engineered vascular grafts (TEVGs) were implanted into Sprague Dawley rats for 5 days to observe SMC population and collagen production. TEVGs were fabricated using a combined computational and experimental approach that varied the ratio of gelatin:polycaprolactone to be either compliance matched or twice as stiff as rat aorta (hypocompliant). TGFß2 concentrations of 0, 10, 100 ng/mg were added to both graft types (n = 3 in each group) and imaged in vivo using ultrasound. Histological markers (SMC, macrophage, collagen, and elastin) were evaluated following explanation at 5 days. In vivo ultrasound showed that compliance-matched TEVGs became stiffer as TGFß2 increased (100 ng/mg TEVGs compared to rat aorta, p < 0.01), while all hypocompliant grafts remained stiffer than control rat aorta. In vivo velocity and diameter were also not significantly different than control vessels. The compliance-matched 10 ng/mg group had an elevated SMC signal (myosin heavy chain) compared to the 0 and 100 ng/mg grafts (p = 0.0009 and 0.0006). Compliance-matched TEVGs containing 100 ng/mg TGFß2 had an increase in collagen production (p < 0.01), general immune response (p < 0.05), and a decrease in SMC population to the 0 and 10 ng/mg groups. All hypocompliant groups were found to be similar, suggesting a lower rate of TGFß2 release in these TEVGs. Our results suggest that TGFß2 can modulate in vivo SMC phenotype over an acute implantation period, which is consistent with our prior in vitro work. To the author's knowledge, this is the first in vivo rat study that evaluates a TGFß2-eluting TEVG. Impact statement TGFß2 affects the SMCs in a vascular graft.

Lymphangiogenesis and angiogenesis: concurrence and/or dependence? Studies in inbred mouse strains.

Genetic background significantly affects angiogenesis in mice. However, lymphangiogenic response to growth factors (GFs) in different strains has not been studied. We report constitutive expression of corneal lymphatics that extends beyond the limits of normal limbal vessels. In untreated corneas, the total number (P=0.006), the number above blood vessels (P=10(-8)), and the area of preexisting lymphatics (P=0.007) were significantly higher in C57BL/6 than in BALB/c mice. Normal corneas of three other strains, the nu/nu, 129E, and Black Swiss mice, showed in most parameters intermediate phenotypes. FGF-2(-/-) mice showed significantly less preexisting lymphatics than control (P=0.009), which suggests a role for this GF in lymphatic development. VEGF-A-induced corneal lymphangiogenic response was significantly higher in BALB/c mice (P=0.03), but it did not differ significantly in C57BL/6 mice, when compared to PBS-implanted control. FGFR-3 expression was higher in C57BL/6 than BALB/c mice, which suggests GF-receptor heterogeneity as a possible explanation for strain-dependent differences. The heterogeneity of preexisting lymphatic vessels in the limbal area significantly correlated with the extent of corneal lymphangiogenesis (VEGF-A: r=0.7, P=0.01; FGF-2: r=0.96, P=10(-5)) in BALB/c but not in C57BL/6 mice. Removal of conjunctival lymphatics did not affect GF-induced lymphangiogenesis. This work introduces physiological expression of lymphatics without blood vessels, which indicates that angiogenesis and lymphangiogenesis, even though intricately related, may occur independently. Furthermore, we show strain-dependence of normal and GF-induced lymphangiogenesis. These differences may affect disease development in various strains.

n-6 Linoleic Acid Induces Epigenetics Alterations Associated with Colonic Inflammation and Cancer.

The farnesoid-X-receptor (FXR) protects against inflammation and cancer of the colon through maintenance of intestinal bile acid (BA) homeostasis. Conversely, higher levels of BA and cyclooxygenase-2 (COX-2) are risk factors for inflammation and cancer of the colon. In the United States, n-6 linoleic acid (LA) is the most commonly used dietary vegetable fat. Metabolism of n-6 fatty acids has been linked to a higher risk of intestinal cancer. The objectives of this study were to investigate in colonic mucosa the effects of a high-fat diet rich in LA (n-6HFD) on CpG methylation of Fxr and prostaglandin-endoperoxide synthase-2 (Ptsg-2) genes, and the impact on the expression of tumor suppressor adenomatous polyposis Coli (Apc) and proliferative cyclin D1 (Ccnd1) genes. Weaned C57BL/6J male mice were fed for 6 weeks either an n-6HFD containing 44% energy (44%E) from 22% safflower oil (SO, 76% LA by weight) or a 13% energy (13%E) control diet (Control) from SO (5% by weight). Mice fed the n-6HFD had reduced (60%) Fxr promoter CpG methylation and increased (~50%) Fxr mRNA. The expression of FXR-target ileal bile acid-binding protein (Ibabp), small heterodimer protein (Shp), and anti-inflammatory peroxisome proliferator-activated-γ1 genes was increased. The n-6HFD reduced Ptgs-2 CpG methylation, increased the expression of Cox-2, and increased Apc CpG methylation in colonic mucosa. Accordingly, reduced expression of Apc was coupled to accumulation of c-JUN and Ccnd1, respectively cofactor and gene targets for the ß-catenin/Wnt signaling pathway. Finally, the n-6HFD reduced the expression of histone deacetylase-1 while favoring the accumulation of acetylated histone 3. We conclude that an n-6HFD epigenetically modifies Fxr, leading to the activation of downstream factors that participate in BA homeostasis. However, epigenetic activation of Ptsg-2 coupled with silencing of Apc and accumulation of C-JUN and Ccnd1 may increase the risk of inflammation and cancer of the colon.

Mediterranean Diet: Prevention of Colorectal Cancer.

Colorectal cancer (CRC) is the third most common cancer diagnosis and the second and third leading cause of cancer mortality in men and women, respectively. However, the majority of CRC cases are the result of sporadic tumorigenesis via the adenoma-carcinoma sequence. This process can take up to 20 years, suggesting an important window of opportunity exists for prevention such as switching toward healthier dietary patterns. The Mediterranean diet (MD) is a dietary pattern associated with various health benefits including protection against cardiovascular disease, diabetes, obesity, and various cancers. In this article, we review publications available in the PubMed database within the last 10 years that report on the impact of a MD eating pattern on prevention of CRC. To assist the reader with interpretation of the results and discussion, we first introduce indexes and scoring systems commonly used to experimentally determine adherence to a MD, followed by a brief introduction of the influence of the MD pattern on inflammatory bowel disease, which predisposes to CRC. Finally, we discuss key biological mechanisms through which specific bioactive food components commonly present in the MD are proposed to prevent or delay the development of CRC. We close with a discussion of future research frontiers in CRC prevention with particular reference to the role of epigenetic mechanisms and microbiome related to the MD eating pattern.

Functional Changes in the Gut Microbiome Contribute to Transforming Growth Factor ß-Deficient Colon Cancer.

Colorectal cancer (CRC) is one of the most treatable cancers, with a 5-year survival rate of ~64%, yet over 50,000 deaths occur yearly in the United States. In 15% of cases, deficiency in mismatch repair leads to null mutations in transforming growth factor ß (TGF-ß) type II receptor, yet genotype alone is not responsible for tumorigenesis. Previous work in mice shows that disruptions in TGF-ß signaling combined with Helicobacter hepaticus cause tumorigenesis, indicating a synergistic effect between genotype and microbial environment. Here, we examine functional shifts in the gut microbiome in CRC using integrated -omics approaches to untangle the role of host genotype, inflammation, and microbial ecology. We profile the gut microbiome of 40 mice with/without deficiency in TGF-ß signaling from a Smad3 (mothers against decapentaplegic homolog-3) knockout and with/without inoculation with H. hepaticus. Clear functional differences in the microbiome tied to specific bacterial species emerge from four pathways related to human colon cancer: lipopolysaccharide (LPS) production, polyamine synthesis, butyrate metabolism, and oxidative phosphorylation (OXPHOS). Specifically, an increase in Mucispirillum schaedleri drives LPS production, which is associated with an inflammatory response. We observe a commensurate decrease in butyrate production from Lachnospiraceae bacterium A4, which could promote tumor formation. H. hepaticus causes an increase in OXPHOS that may increase DNA-damaging free radicals. Finally, multiple bacterial species increase polyamines that are associated with colon cancer, implicating not just diet but also the microbiome in polyamine levels. These insights into cross talk between the microbiome, host genotype, and inflammation could promote the development of diagnostics and therapies for CRC. IMPORTANCE Most research on the gut microbiome in colon cancer focuses on taxonomic changes at the genus level using 16S rRNA gene sequencing. Here, we develop a new methodology to integrate DNA and RNA data sets to examine functional shifts at the species level that are important to tumor development. We uncover several metabolic pathways in the microbiome that, when perturbed by host genetics and H. hepaticus inoculation, contribute to colon cancer. The work presented here lays a foundation for improved bioinformatics methodologies to closely examine the cross talk between specific organisms and the host, important for the development of diagnostics and pre/probiotic treatment.

Genistein Prevents BRCA1 CpG Methylation and Proliferation in Human Breast Cancer Cells with Activated Aromatic Hydrocarbon Receptor.

Background: Previous studies have suggested a causative role for agonists of the aromatic hydrocarbon receptor (AhR) in the etiology of breast cancer 1, early-onset (BRCA-1)-silenced breast tumors, for which prospects for treatment remain poor. Objectives: We investigated the regulation of BRCA1 by the soy isoflavone genistein (GEN) in human estrogen receptor α (ERα)-positive Michigan Cancer Foundation-7 (MCF-7) and ERα-negative sporadic University of Arizona Cell Culture-3199 (UACC-3199) breast cancer cells, respectively, with inducible and constitutively active AhR. Methods: In MCF-7 cells, we analyzed the dose- and time-dependent effects of GEN and (-)-epigallocatechin-3-gallate (EGCG) control, selected as prototype dietary DNA methyltransferase (DNMT) inhibitors, on BRCA-1 expression after AhR activation with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and in TCDD-washout experiments. We compared the effects of GEN and EGCG on BRCA1 cytosine-phosphate-guanine (CpG) methylation and cell proliferation. Controls for DNA methylation and proliferation were changes in expression of DNMT-1, cyclin D1, and p53, respectively. In UACC-3199 cells, we compared the effects of GEN and α-naphthoflavone (αNF; 7,8-benzoflavone), a synthetic flavone and AhR antagonist, on BRCA1 expression and CpG methylation, cyclin D1, and cell growth. Finally, we examined the effects of GEN and αNF on BRCA1, AhR-inducible cytochrome P450 (CYP)-1A1 (CYP1A1) and CYP1B1, and AhR mRNA expression. Results: In MCF-7 cells, GEN exerted dose- and time-dependent preventative effects against TCDD-dependent downregulation of BRCA-1. After TCDD washout, GEN rescued BRCA-1 protein expression while reducing DNMT-1 and cyclin D1. GEN and EGCG reduced BRCA1 CpG methylation and cell proliferation associated with increased p53. In UACC-3199 cells, GEN reduced BRCA1 and estrogen receptor-1 (ESR1) CpG methylation, cyclin D1, and cell growth while inducing BRCA-1 and CYP1A1. Conclusions: Results suggest preventative effects for GEN and EGCG against BRCA1 CpG methylation and downregulation in ERα-positive breast cancer cells with activated AhR. GEN and flavone antagonists of AhR may be useful for reactivation of BRCA1 and ERα via CpG demethylation in ERα-negative breast cancer cells harboring constitutively active AhR.

Elimination of colon cancer in germ-free transforming growth factor beta 1-deficient mice.

Patients with ulcerative colitis are at risk for colon cancer and frequently have microsatellite instability,which, in turn, is usually associated with inactivation of transforming growth factor (TGF) beta signaling. TGF-beta1 deficiency in mice can lead to colon cancer that is preceded by precancerous lesions having submucosal inflammation and hyperplastic crypts. Germ-free TGF-beta1-deficient mice are free of inflammation, hyperplasia, and cancer, but when reintroduced into a Helicobacter hepaticus-containing specific pathogen-free room, these lesions reappear. Because adenoma/carcinoma but not inflammation/hyperplasia is dependent on the genetic backgrounds tested, colitis is required, but not sufficient, for carcinogenesis. This animal model should provide insight into the protective role of TGF-beta1 in early stages of ulcerative colitis-associated human colon cancer.

TGFß2 differentially modulates smooth muscle cell proliferation and migration in electrospun gelatin-fibrinogen constructs.

A main goal of tissue engineering is the development of scaffolds that replace, restore and improve injured tissue. These scaffolds have to mimic natural tissue, constituted by an extracellular matrix (ECM) support, cells attached to the ECM, and signaling molecules such as growth factors that regulate cell function. In this study we created electrospun flat sheet scaffolds using different compositions of gelatin and fibrinogen. Smooth muscle cells (SMCs) were seeded on the scaffolds, and proliferation and infiltration were evaluated. Additionally, different concentrations of Transforming Growth Factor-beta2 (TGFß2) were added to the medium with the aim of elucidating its effect on cell proliferation, migration and collagen production. Our results demonstrated that a scaffold with a composition of 80% gelatin-20% fibrinogen is suitable for tissue engineering applications since it promotes cell growth and migration. The addition of TGFß2 at low concentrations (≤ 1 ng/ml) to the culture medium resulted in an increase in SMC proliferation and scaffold infiltration, and in the reduction of collagen production. In contrast, TGFß2 at concentrations >1 ng/ml inhibited cell proliferation and migration while stimulating collagen production. According to our results TGFß2 concentration has a differential effect on SMC function and thus can be used as a biochemical modulator that can be beneficial for tissue engineering applications.

The neural crest is contiguous with the cardiac conduction system in the mouse embryo: a role in induction?

In this study we present data on the spatial relationship between neural crest-derived cells (NCC) and the specialized cardiac conduction system (CCS) in the developing murine heart. Using Wnt1-Cre/R26R conditional reporter mice that express beta-galactosidase from ROSA26 upon Cre-mediated recombination, two populations of NCC are seen: one migrates through the arterial pole and contributes to the bundle branches, whereas the second population enters by way of the venous pole and provides cells to the sinoatrial and atrioventricular node areas. The CCS/ lacZ construct is found in the myocardium of the early embryonic heart and afterward only persists in the definitive CCS and is acknowledged as a reporter for the developing conduction system. The contiguous expression of both reporters is suggestive for a potential role of cardiac NCC in the induction of the final differentiation of the CCS.

Cardiac development and physiology are modulated by FGF2 in an isoform- and sex-specific manner.

The low molecular weight isoform (Lo) of fibroblast growth factor 2 (FGF2) has distinct functions from the high molecular weight isoforms (Hi) of FGF2 in the adult stressed heart. However, the specific roles of these isoforms in the unstressed heart were not examined. We investigated whether the FGF2 isoforms modulate cardiac development and physiology in isoform- and sex-specific manners. Young adult male and female mice that were deficient in either Hi FGF2 (Hi KO) or Lo FGF2 (Lo KO) underwent echocardiographic analysis and were compared to their wildtype (WT) counterparts. By comparison to WT cohorts, female Lo KO hearts display a 33% larger left ventricular (LV) volume and smaller LV mass and wall thickness. Mitral valve flow measurements from these hearts reveal that the early wave to atrial wave ratio (E/A) is higher, the deceleration time is 30% shorter and the mitral valve E-A velocity time integral is reduced by 20% which is consistent with a restrictive filling pattern. The female Hi KO hearts do not demonstrate any significant abnormality. In male Hi KO mice the cardiac output from the LV is 33% greater and the fractional shortening is 29% greater, indicating enhanced systolic function, while in male Lo KO hearts we observe a smaller E/A ratio and a prolonged isovolumic relaxation time, consistent with an impaired relaxation filling pattern. We conclude that the developmental and physiological functions of FGF2 isoforms in the unstressed heart are isoform-specific and non-redundant and that these roles are modulated by sex.

FGF2 modulates cardiac remodeling in an isoform- and sex-specific manner.

Pathological cardiac hypertrophy and cardiac fibrosis are remodeling events that result in mechanical stiffness and pathophysiological changes of the myocardium. Both humans and animal models display a sexual dimorphism where females are more protected from pathological remodeling. Fibroblast growth factor 2 (FGF2) mediates cardiac hypertrophy, cardiac fibrosis and protection against cardiac injury and is made in high molecular weight and low molecular weight isoforms (Hi FGF2 and Lo FGF2, respectively). Although some light has been shed on isoform-specific functions in cardiac pathophysiology, their roles in pathologic cardiac remodeling have yet to be determined. We tested the hypothesis that Lo FGF2 and Hi FGF2 modulate pathological cardiac remodeling in an isoform-specific manner. Young adult male and female mice between 8-12 weeks of age of mixed background that were deficient in either Hi FGF2 or Lo FGF2 (Hi KO or Lo KO, respectively) were subjected to daily injections of isoproterenol (Iso) for four days after which their hearts were compared to wildtype cohorts. Post-Iso treatment, female Lo KO hearts don't exhibit significant differences in their hypertrophic and fibrotic response, while female Hi KO hearts present with a blunted hypertrophic response. In male animals Lo KO hearts present with an exacerbated fibrotic response and increased alpha-smooth muscle actin protein expression while Hi KO hearts present with a blunted fibrotic response and increased atrial natriuretic factor protein expression Thus, in female hearts Hi FGF2 mediate cardiac hypertrophy while in male hearts Lo FGF2 and Hi FGF2 display an antithetical role in cardiac fibrosis where Lo FGF2 is protective while Hi FGF2 is damaging. In conclusion, cardiac remodeling following catecholamine overactivation is modulated by FGF2 in isoform- and sex-specific manners.

Biological functions of the low and high molecular weight protein isoforms of fibroblast growth factor-2 in cardiovascular development and disease.

Fibroblast growth factor 2 (FGF2) consists of multiple protein isoforms (low molecular weight, LMW, and high molecular weight, HMW) produced by alternative translation from the Fgf2 gene. These protein isoforms are localized to different cellular compartments, indicating unique biological activity. FGF2 isoforms in the heart have distinct roles in many pathological circumstances in the heart including cardiac hypertrophy, ischemia-reperfusion injury, and atherosclerosis. These studies suggest distinct biological activities of FGF2 LMW and HMW isoforms both in vitro and in vivo. Yet, due to the limitations that only the recombinant FGF2 LMW isoform is readily available and that the FGF2 antibody is nonspecific with regards to its isoforms, much remains to be determined regarding the role(s) of the FGF2 LMW and HMW isoforms in cellular behavior and in cardiovascular development and pathophysiology. This review summarizes the activities of LMW and HMW isoforms of FGF2 in cardiovascular development and disease.

Gene targeted ablation of high molecular weight fibroblast growth factor-2.

Fibroblast growth factor-2 (FGF2) is produced as high molecular weight isoforms (HMW) and a low molecular weight isoform (LMW) by means of alternative usage of translation start sites in a single Fgf2 mRNA. Although the physiological function of FGF2 and FGF2 LMW has been investigated in myocardial capillarogenesis during normal cardiac growth, the role of FGF2 HMW has not been determined. Here, we report the generation of FGF2 HMW-deficient mice in which FGF2 HMW isoforms are ablated by the Tag-and-Exchange gene targeting technique. These mice are normal and fertile with normal fecundity, and have a normal life span. Histological, immunohistochemical, and morphometric analyses indicate normal myocardial architecture, blood vessel, and cardiac capillary density in young adult FGF2 HMW-deficient mice. These mice along with the FGF2- and FGF2 LMW-deficient mice that we have generated previously will be very useful for elucidating the differential functions of FGF2 isoforms in pathophysiology of cardiovascular diseases.

Altered renin synthesis and secretion in the kidneys of heterozygous mice with a null mutation in the TGF-beta(2) gene.

Transforming growth factors beta (TGF-betas) are peptides involved in autocrine and paracrine control of cell growth and differentiation. In the kidneys, TGF-beta(2) has been shown to localize specifically in renin-producing cells in various conditions stimulating the renin response. To test in vivo the functional role of TGF-beta(2), the renin response was investigated in mice heterozygous for a null mutation of the TGF-beta(2) gene, which had a twofold reduction in the amount of TGF-beta(2) mRNA. Although the increase in plasma renin concentration triggered by dehydration was not different from wild-type mice, renal renin mRNA and protein levels were higher in mutant mice under hydrated or dehydrated conditions. These data suggest that TGF-beta(2) exerts an inhibitory effect on renin synthesis and release from the juxtaglomerular apparatuses.