hCG stimulates angiogenic signals in lymphatic endothelial and circulating angiogenic cells.

Human chorionic gonadotropin (hCG) has long been associated with the initiation and maintenance of pregnancy, where angiogenesis plays an important role. However, the function of hCG in angiogenesis and the recruitment of vascular active cells are not fully understood. In this study, the role of hCG and its receptor in circulating angiogenic and human endothelial cells, including lymphatic, uterine microvascular, and umbilical vein endothelial cells, was examined. Immunohistochemistry and immunoblot analysis were used to detect LH/hCG receptor expression and the expression of hCG-induced angiogenic molecules. HIF-1α was determined via ELISA and downstream molecules, such as CXCL12 and CXCR4, via real-time PCR. Chemotaxis was analyzed using Boyden chambers. Our results show that the LH/hCG receptor was present in all tested cells. Furthermore, hCG was able to stimulate LH/hCG-receptor-specific migration in a dose-dependent fashion and induce key angiogenic molecules, including HIF-1α, CXCL12, and CXCR4. In conclusion, our findings underscore the importance of hCG as one of the first angiogenic molecules produced by the conceptus. hCG itself alters endothelial motility, recruitment, and expression of pro-angiogenic molecules and may therefore play an important role in vascular adaption during implantation and early placental formation.

Early and late response-to-injury in patients undergoing transradial coronary angiography: arterial remodeling in smokers.

OBJECTIVES: To investigate the effect of smoking on vascular response to transradial coronary angiography (TCA). BACKGROUND: Cigarette smoking is the most important modifiable cardiovascular risk factor associated with endothelial dysfunction. METHODS: Radial artery flow-mediated vasodilation (RA-FMD), local stiffness (fractional diameter change), intima-media thickness (IMT), luminal and external arterial diameter were measured in 40 current smokers (CS) and former smokers (FS) at 6-14 months at the site of previous TCA and contralateral control artery. Vascular regenerative capacity was studied as chemotactic cell migration in vitro and ex vivo (n=10) and the time course of endothelial functional recovery following RA-FMD up to 72 h after TCA (n=10). RESULTS: At 10 ± 3 months after TCA, subjects exhibited significant local stiffening and increased IMT as compared to the control arm. These late structural changes were significantly more pronounced in CS as compared to FS. IMT thickening correlated with packyears, number of daily cigarettes, and inversely with RA-FMD. Nitric oxide synthase (NOS)-dependent chemotaxis of CS' circulating angiogenic cells was impaired. Ex vivo incubation of endothelial cells with CS' plasma inhibited NOS-dependent endothelial wound closure and chemotaxis. In vivo, TCA acutely decreased RA-FMD. At 24 h, RA-FMD had recovered in FS but remained impaired at 24 h and only recovered at 48 h in CS. CONCLUSION: In active smokers, transradial coronary angiography is associated with delayed early recovery from transient endothelial dysfunction, decreased NOS-dependent vascular regeneration, and late arterial remodeling pointing towards potential harmful effects of transradial coronary angiography on vascular function in distinct subsets of patients.

Two isoforms of Sister-Of-Mammalian Grainyhead have opposing functions in endothelial cells and in vivo.

OBJECTIVE: Sister-of-Mammalian Grainyhead (SOM) is a member of the Grainyhead family of transcription factors. In humans, 3 isoforms are derived from differential first exon usage and alternative splicing and differ only in their N terminal domain. SOM2, the only variant also present in mouse, induces endothelial cell migration and protects against apoptosis. The functions of the human specific isoforms SOM1 and SOM3 have not yet been investigated. Therefore we wanted to elucidate their functions in endothelial cells. APPROACH AND RESULTS: Overexpression of SOM1 in primary human endothelial cells induced migration, phosphorylation of Akt1 and endothelial nitric oxide synthase, and protected against apoptosis, whereas SOM3 had opposite effects; isoform-specific knockdowns confirmed the disparate effects on apoptosis. After reporter assays demonstrated that both are active transcription factors, microarray analyses revealed that they induce different target genes, which could explain the different cellular effects. Overexpression of SOM3 in zebrafish embryos resulted in increased lethality and severe deformations, whereas SOM1 had no deleterious effect. CONCLUSIONS: Our data demonstrate that the splice variant-derived isoforms SOM1 and SOM3 induce opposing effects in primary human endothelial cells and in a whole animal model, most likely through the induction of different target genes.

The transcription factor Grainyhead like 3 (GRHL3) affects endothelial cell apoptosis and migration in a NO-dependent manner.

Migratory capacity and resistance to apoptosis are crucial for proper endothelial function. In a screen for anti-apoptotic genes in a breast cancer cell line, we identified Grainyhead like 3 (GRHL3). Therefore, the aim of our study was to investigate whether GRHL3 is expressed in endothelial cells and moreover, to determine its role in migration, apoptosis and senescence. GRHL3 is expressed in human endothelial cells. GRHL3 is required for endothelial cell migration. The underlying mechanism is independent of vascular endothelial growth factor. GRHL3 induces Akt and endothelial nitric oxide synthase phosphorylation and its expression is increased by physiological concentrations of nitric oxide. Nitric oxide dependent migration is completely dependent on GRHL3 expression. Moreover, GRHL3 inhibits apoptosis of endothelial cells in an eNOS-dependent manner. Thus, loss of GRHL3 may result in endothelial dysfunction in vivo. One may consider new therapeutic strategies with the aim to conserve GRHL3 expression in the vasculature.

Downregulation of mitochondrial telomerase reverse transcriptase induced by H2O2 is Src kinase dependent.

Telomerase with its catalytic subunit telomerase reverse transcriptase (TERT) prevents telomere erosion in the nucleus. In addition, telomerase has also telomere-independent functions in protection from apoptosis. Unexpectedly, TERT was found in the mitochondria. However, its regulation in this organelle is completely unknown. Here, we demonstrate that mitochondrial TERT is downregulated by exposure to H(2)O(2) in primary human endothelial cells. This depletion is dependent on the Src phosphorylation site within TERT, tyrosine 707. In accordance with this finding, we also detected Src in the mitochondria and demonstrated that Src is activated upon H(2)O(2) treatment. This regulation of mitochondrial TERT is reminiscent of the situation in the nucleus from where TERT is exported under conditions of oxidative stress in a Src kinase dependent manner. In addition, Akt1 was also found in the mitochondria and H(2)O(2) treatment led to reduced active Akt1 in these organelles, suggesting that similar regulatory mechanisms operate in mitochondria and the nucleus.

Nuclear redox signaling.

Reactive oxygen species have been described to modulate proteins within the cell, a process called redox regulation. However, the importance of compartment-specific redox regulation has been neglected for a long time. In the early 1980s and 1990s, many in vitro studies introduced the possibility that nuclear redox signaling exists. However, the functional relevance for that has been greatly disregarded. Recently, it has become evident that nuclear redox signaling is indeed one important signaling mechanism regulating a variety of cellular functions. Transcription factors, and even kinases and phosphatases, have been described to be redox regulated in the nucleus. This review describes several of these proteins in closer detail and explains their functions resulting from nuclear localization and redox regulation. Moreover, the redox state of the nucleus and several important nuclear redox regulators [Thioredoxin-1 (Trx-1), Glutaredoxins (Grxs), Peroxiredoxins (Prxs), and APEX nuclease (multifunctional DNA-repair enzyme) 1 (APEX1)] are introduced more precisely, and their necessity for regulation of transcription factors is emphasized.

Nuclear protein tyrosine phosphatase Shp-2 is one important negative regulator of nuclear export of telomerase reverse transcriptase.

Aging is one major risk factor for numerous diseases. The enzyme telomerase reverse transcriptase (TERT) plays an important role for aging and apoptosis. Previously, we demonstrated that inhibition of oxidative stress-induced Src kinase family-dependent nuclear export of TERT results in delayed replicative senescence and reduced apoptosis sensitivity. Therefore, the aim of this study was to investigate mechanisms inhibiting nuclear export of TERT. First, we demonstrated that H2O2-induced nuclear export of TERT was abolished in Src, Fyn, and Yes-deficient embryonic fibroblasts. Next, we wanted to identify one potential negative regulator of this export process. One candidate is the protein tyrosine phosphatase Shp-2 (Shp-2), which can counteract activities of the Src kinase family. Indeed, Shp-2 was evenly distributed between the nucleus and cytosol. Nuclear Shp-2 associates with TERT in endothelial cells and dissociates from TERT prior to its nuclear export. Overexpression of Shp-2 wt inhibited H2O2-induced export of TERT. Overexpression of the catalytically inactive, dominant negative Shp-2 mutant (Shp-2(C459S)) reduced endogenous as well as overexpressed nuclear TERT protein and telomerase activity, whereas it had no influence on TERT(Y707F). Binding of TERT(Y707F) to Shp-2 is reduced compared with TERTwt. Ablation of Shp-2 expression led only to an increased tyrosine phosphorylation of TERTwt, but not of TERT(Y707F). Moreover, reduced Shp-2 expression decreased nuclear telomerase activity, whereas nuclear telomerase activity was increased in Shp-2-overexpressing endothelial cells. In conclusion, Shp-2 retains TERT in the nucleus by regulating tyrosine 707 phosphorylation.

Gene trapping identifies a putative tumor suppressor and a new inducer of cell migration.

Tumor necrosis factor alpha (TNFalpha) is a pleiotropic cytokine involved in apoptotic cell death, cellular proliferation, differentiation, inflammation, and tumorigenesis. In tumors it is secreted by tumor associated macrophages and can have both pro- and anti-tumorigenic effects. To identify genes regulated by TNFalpha, we performed a gene trap screen in the mammary carcinoma cell line MCF-7 and recovered 64 unique, TNFalpha-induced gene trap integration sites. Among these were the genes coding for the zinc finger protein ZC3H10 and for the transcription factor grainyhead-like 3 (GRHL3). In line with the dual effects of TNFalpha on tumorigenesis, we found that ZC3H10 inhibits anchorage independent growth in soft agar suggesting a tumor suppressor function, whereas GRHL3 strongly stimulated the migration of endothelial cells which is consistent with an angiogenic, pro-tumorigenic function.