Intravenous delivery of the plasma fraction of stored packed erythrocytes promotes pancreatic cancer growth in immunocompetent mice.

BACKGROUND: Perioperative blood transfusion in pancreatic cancer patients has been linked to decreased survival; however, a causal mechanism has not been determined. During the processing and storage of packed erythrocytes, biologically active molecules accumulated in the acellular plasma fraction; therefore, the authors hypothesized that the plasma fraction of stored packed erythrocytes promoted tumor progression. METHODS: Proliferation and migration of murine pancreatic cancer and control cells were determined in vitro in response to the plasma fraction from leukocyte and nonleukocyte-reduced fresh versus stored packed erythrocytes. Last, an immunocompetent murine model was used to assess the effect of the plasma fraction of stored and processed packed erythrocytes on pancreatic cancer progression. RESULTS: Incubation of pancreatic cancer cells with the plasma fraction of packed erythrocytes increased proliferation and migration. Intravenous delivery of the acellular plasma fraction to mice with pancreatic cancer significantly increased the tumor weight in both leukocyte-reduced and nonleukocyte-reduced packed-erythrocyte groups (P<.01), although tumor growth and morbidity were greatest in the nonleukocyte-reduced group. CONCLUSIONS: The plasma fraction of stored packed erythrocytes promoted murine pancreatic cancer proliferation and migration in vitro and when administered intravenously, significantly augmented pancreatic cancer growth in immunocompetent mice.

Loss of fibulin-5 binding to beta1 integrins inhibits tumor growth by increasing the level of ROS.

Tumor survival depends in part on the ability of tumor cells to transform the surrounding extracellular matrix (ECM) into an environment conducive to tumor progression. Matricellular proteins are secreted into the ECM and impact signaling pathways that are required for pro-tumorigenic activities such as angiogenesis. Fibulin-5 (Fbln5) is a matricellular protein that was recently shown to regulate angiogenesis; however, its effect on tumor angiogenesis and thus tumor growth is currently unknown. We report that the growth of pancreatic tumors and tumor angiogenesis is suppressed in Fbln5-null (Fbln5(-/-)) mice compared with wild-type (WT) littermates. Furthermore, we observed an increase in the level of reactive oxygen species (ROS) in tumors grown in Fbln5(-/-) animals. Increased ROS resulted in elevated DNA damage, increased apoptosis of endothelial cells within the tumor, and represented the underlying cause for the reduction in angiogenesis and tumor growth. In vitro, we identified a novel pathway by which Fbln5 controls ROS production through a mechanism that is dependent on beta1 integrins. These results were validated in Fbln5(RGE/RGE) mice, which harbor a point mutation in the integrin-binding RGD motif of Fbln5, preventing its interaction with integrins. Tumor growth and angiogenesis was reduced in Fbln5(RGE/RGE) mice, however treatment with an antioxidant rescued angiogenesis and elevated tumor growth to WT levels. These findings introduce a novel function for Fbln5 in the regulation of integrin-induced ROS production and establish a rationale for future studies to examine whether blocking Fbln5 function could be an effective anti-tumor strategy, alone or in combination with other therapies.

Fibulin-5, an integrin-binding matricellular protein: its function in development and disease.

Interactions between the extracellular matrix (ECM) and cells are critical in embryonic development, tissue homeostasis, physiological remodeling, and tumorigenesis. Matricellular proteins, a group of ECM components, mediate cell-ECM interactions. One such molecule, Fibulin-5 is a 66-kDa glycoprotein secreted by various cell types, including vascular smooth muscle cells (SMCs), fibroblasts, and endothelial cells. Fibulin-5 contributes to the formation of elastic fibers by binding to structural components including tropoelastin and fibrillin-1, and to cross-linking enzymes, aiding elastic fiber assembly. Mice deficient in the fibulin-5 gene (Fbln5) exhibit systemic elastic fiber defects with manifestations of loose skin, tortuous aorta, emphysematous lung and genital prolapse. Although Fbln5 expression is down-regulated after birth, following the completion of elastic fiber formation, expression is reactivated upon tissue injury, affecting diverse cellular functions independent of its elastogenic function. Fibulin-5 contains an evolutionally conserved arginine-glycine-aspartic acid (RGD) motif in the N-terminal region, which mediates binding to a subset of integrins, including alpha5beta1, alphavbeta3, and alphavbeta5. Fibulin-5 enhances substrate attachment of endothelial cells, while inhibiting migration and proliferation in a cell type- and context-dependent manner. The antagonistic function of fibulin-5 in angiogenesis has been demonstrated in vitro and in vivo; fibulin-5 may block angiogenesis by inducing the anti-angiogenic molecule thrompospondin-1, by antagonizing VEGF(165)-mediated signaling, and/or by antagonizing fibronectin-mediated signaling through directly binding and blocking the alpha5beta1 fibronectin receptor. The overall effect of fibulin-5 on tumor growth depends on the balance between the inhibitory property of fibulin-5 on angiogenesis and the direct effect of fibulin-5 on proliferation and migration of tumor cells. However, the effect of tumor-derived versus host microenvironment-derived fibulin-5 remains to be evaluated.

Interaction of Gata4 and Gata6 with Tbx5 is critical for normal cardiac development.

Congenital heart disease is the most common type of birth defect with an incidence of 1%. Previously, we described a point mutation in GATA4 that segregated with cardiac defects in a family with autosomal dominant disease. The mutation (G296S) exhibited biochemical deficits and disrupted a novel interaction between Gata4 and Tbx5. To determine if Gata4 and Tbx5 genetically interact in vivo, we generated mice heterozygous for both alleles. We found that nearly 100% of mice heterozygous for Gata4 and Tbx5 were embryonic or neonatal lethal and had complete atrioventricular (AV) septal defects with a single AV valve and myocardial thinning. Consistent with this phenotype, Gata4 and Tbx5 are co-expressed in the developing endocardial cushions and myocardium. In mutant embryos, cardiomyocyte proliferation deficits were identified compatible with the myocardial hypoplasia. Similar to Gata4, Gata6 and Tbx5 are co-expressed in the embryonic heart, and the transcription factors synergistically activate the atrial natiuretic factor promoter. We demonstrate a genetic interaction between Gata6 and Tbx5 with an incompletely penetrant phenotype of neonatal lethality and thin myocardium. Gene expression analyses were performed on both sets of compound heterozygotes and demonstrated downregulation of alpha-myosin heavy chain only in Gata4/Tbx5 heterozygotes. These findings highlight the unique genetic interactions of Gata4 and Gata6 with Tbx5 for normal cardiac morphogenesis in vivo.

Screening and biochemical analysis of GATA4 sequence variations identified in patients with congenital heart disease.

Few known monogenic causes of non-syndromic congenital heart disease (CHD) have been identified. Mutations in NKX2.5 were initially implicated in familial cases of cardiac septal defects and subsequently, functionally significant NKX2.5 mutations were found in diverse forms of non-syndromic CHD. Similarly, mutations in GATA4, which encodes a cardiac transcription factor, were first identified in familial cases of cardiac septal defects. We hypothesize that individuals with non-syndromic CHD may harbor GATA4 mutations and that these mutations alter the biochemical properties of the protein. The coding region encompassing the six exons of GATA4 was screened in a study population of 157 patients with CHD. We identified several sequence variations in GATA4. We tested these novel sequence variations that altered evolutionarily conserved amino acids and other previously reported GATA4 mutations in various biochemical assays. The novel sequence variations had no biochemical deficits while a previously reported, but unstudied, missense mutation in GATA4 (S52F) functioned as a hypomorph in transactivation assays. We did not identify any novel GATA4 mutations in our patient population with non-syndromic CHD. Consistent with previous findings, GATA4 mutations that result in deficits in transactivation ability are consistently associated with CHD suggesting that normal transactivation properties of GATA4 are required for proper cardiac development.

Mutations in NOTCH1 cause aortic valve disease.

Calcification of the aortic valve is the third leading cause of heart disease in adults. The incidence increases with age, and it is often associated with a bicuspid aortic valve present in 1-2% of the population. Despite the frequency, neither the mechanisms of valve calcification nor the developmental origin of a two, rather than three, leaflet aortic valve is known. Here, we show that mutations in the signalling and transcriptional regulator NOTCH1 cause a spectrum of developmental aortic valve anomalies and severe valve calcification in non-syndromic autosomal-dominant human pedigrees. Consistent with the valve calcification phenotype, Notch1 transcripts were most abundant in the developing aortic valve of mice, and Notch1 repressed the activity of Runx2, a central transcriptional regulator of osteoblast cell fate. The hairy-related family of transcriptional repressors (Hrt), which are activated by Notch1 signalling, physically interacted with Runx2 and repressed Runx2 transcriptional activity independent of histone deacetylase activity. These results suggest that NOTCH1 mutations cause an early developmental defect in the aortic valve and a later de-repression of calcium deposition that causes progressive aortic valve disease.

GATA4 mutations cause human congenital heart defects and reveal an interaction with TBX5.

Congenital heart defects (CHDs) are the most common developmental anomaly and are the leading non-infectious cause of mortality in newborns. Only one causative gene, NKX2-5, has been identified through genetic linkage analysis of pedigrees with non-syndromic CHDs. Here, we show that isolated cardiac septal defects in a large pedigree were linked to chromosome 8p22-23. A heterozygous G296S missense mutation of GATA4, a transcription factor essential for heart formation, was found in all available affected family members but not in any control individuals. This mutation resulted in diminished DNA-binding affinity and transcriptional activity of Gata4. Furthermore, the Gata4 mutation abrogated a physical interaction between Gata4 and TBX5, a T-box protein responsible for a subset of syndromic cardiac septal defects. Conversely, interaction of Gata4 and TBX5 was disrupted by specific human TBX5 missense mutations that cause similar cardiac septal defects. In a second family, we identified a frame-shift mutation of GATA4 (E359del) that was transcriptionally inactive and segregated with cardiac septal defects. These results implicate GATA4 as a genetic cause of human cardiac septal defects, perhaps through its interaction with TBX5.