In vitro pharmacological profile of PHA-022121, a small molecule bradykinin B2 receptor antagonist in clinical development.

PHA-022121 is a novel small molecule bradykinin B2 receptor antagonist, in clinical development for the treatment and prevention of hereditary angioedema attacks. The present study describes the in vitro pharmacological characteristics of PHA-022121 and its active metabolite, PHA-022484 (M2-D). In mammalian cell lines, PHA-022121 and PHA-022484 show high affinity for the recombinant human bradykinin B2 receptor with Ki values of 0.47 and 0.70 nM, respectively, and potent antagonism of the human bradykinin B2 receptor with Kb values of 0.15 and 0.26 nM, respectively (calcium mobilization assay). Antagonist potency at the recombinant cynomolgus monkey bradykinin B2 receptor is similarly high (Kb values of 1.42 and 1.12 nM for PHA-022121 and PHA-022484, respectively), however, potency at rat, mouse, rabbit and dog bradykinin B2 receptors is at least 100-fold lower than the potency at the human receptor for both compounds. In the human umbilical vein contractility assay, both PHA-022121 and PHA-022484 show a potent, surmountable and reversible B2 antagonist activity with pA2 values of 0.35 and 0.47 nM, respectively. The in vitro off-target profile of PHA-022121 and PHA-022484 demonstrates a high degree of selectivity over a wide range of molecular targets, including the bradykinin B1 receptor. It is concluded that PHA-022121 is a novel, low-molecular weight, competitive antagonist of the human bradykinin B2 receptor with high affinity, high antagonist potency, and high selectivity. It is about 20-fold more potent than icatibant at the human bradykinin B2 receptor as assessed using recombinant or endogenously expressed receptors.

Transforming growth factor-ß1 signalling triggers vascular endothelial growth factor resistance and monocyte dysfunction in type 2 diabetes mellitus.

Type 2 diabetes mellitus (T2DM) leads to monocyte dysfunction associated with atherogenesis and defective arteriogenesis. Transforming growth factor (TGF)-ß1, placenta growth factor (PlGF)-1 and vascular endothelial growth factor (VEGF)A play important roles in atherogenesis and arteriogenesis. VEGF-receptor (VEGFR)-mediated monocyte migration is inhibited in T2DM (VEGFA resistance), while TGF-ß1-induced monocyte migration is fully functional. Therefore, we hypothesize that TGF-ß antagonises the VEGFA responses in human monocytes. We demonstrate that monocytes from T2DM patients have an increased migratory response towards low concentrations of TGF-ß1, while PlGF-1/VEGFA responses are mitigated. Mechanistically, this is due to increased expression of type II TGF-ß receptor in monocytes under high-glucose conditions and increased expression of soluble (s)VEGFR1, which is known to interfere with VEGFA signalling. VEGFA resistance in monocytes from T2DM patients can be rescued by either experimental down-regulation of TGF-ß receptor expression in vitro or by functional blocking of TGF-ß signalling using either a TGF-ß receptor kinase inhibitor or a TGF-ß neutralizing antibody. Our data demonstrate that both T2DM and high-glucose potentiate the TGF-ß pathway. TGF-ß signalling impairs VEGFR-mediated responses in T2DM monocytes and in this way contributes to mononuclear cell dysfunction, provide novel insights into T2DM vascular dysfunction.

Full spectrum of clonal haematopoiesis-driver mutations in chronic heart failure and their associations with mortality.

AIMS: Somatic mutations in haematopoietic stem cells can lead to the clonal expansion of mutated blood cells, known as clonal haematopoiesis (CH). Mutations in the most prevalent driver genes DNMT3A and TET2 with a variant allele frequency (VAF) ≥ 2% have been associated with atherosclerosis and chronic heart failure of ischemic origin (CHF). However, the effects of mutations in other driver genes for CH with low VAF (<2%) on CHF are still unknown. METHODS AND RESULTS: Therefore, we analysed mononuclear bone marrow and blood cells from 399 CHF patients by deep error-corrected targeted sequencing of 56 genes and associated mutations with the long-term mortality in these patients (3.95 years median follow-up). We detected 1113 mutations with a VAF ≥ 0.5% in 347 of 399 patients, and only 13% had no detectable CH. Despite a high prevalence of mutations in the most frequently mutated genes DNMT3A (165 patients) and TET2 (107 patients), mutations in CBL, CEBPA, EZH2, GNB1, PHF6, SMC1A, and SRSF2 were associated with increased death compared with the average death rate of all patients. To avoid confounding effects, we excluded patients with DNMT3A-related, TET2-related, and other clonal haematopoiesis of indeterminate potential (CHIP)-related mutations with a VAF ≥ 2% for further analyses. Kaplan-Meier survival analyses revealed a significantly higher mortality in patients with mutations in either of the seven genes (53 patients), combined as the CH-risk gene set for CHF. Baseline patient characteristics showed no significant differences in any parameter including patient age, confounding diseases, severity of CHF, or blood cell parameters except for a reduced number of platelets in patients with mutations in the risk gene set in comparison with patients without. However, carrying a mutation in any of the risk genes remained significant after multivariate cox regression analysis (hazard ratio, 3.1; 95% confidence interval, 1.8-5.4; P < 0.001), whereas platelet numbers did not. CONCLUSIONS: Somatic mutations with low VAF in a distinct set of genes, namely, in CBL, CEBPA, EZH2, GNB1, PHF6, SMC1A, and SRSF2, are significantly associated with mortality in CHF, independently of the most prevalent CHIP-mutations in DNMT3A and TET2. Mutations in these genes are prevalent in young CHF patients and comprise an independent risk factor for the outcome of CHF, potentially providing a novel tool for risk assessment in CHF.

Clonal hematopoiesis, aging, and cardiovascular diseases.

Cardiovascular diseases (CVDs) remain the leading cause of death worldwide. Many studies have provided evidence that both genetic and environmental factors induce atherosclerosis, leading thus to cardiovascular complications. Atherosclerosis is an inflammatory disease, and aging is strongly associated with the development of atherosclerosis. Recent experimental evidence suggests that clonal hematopoiesis (CH) is an emerging cardiovascular risk factor that contributes to the development of atherosclerosis and cardiac dysfunction and exacerbates cardiovascular diseases. CH is caused by somatic mutations in recurrent genes in hematopoietic stem cells, leading to the clonal expansion of mutated blood cell clones. Many of the mutated genes are known in the context of myeloid neoplasms. However, only some individuals carrying CH mutations develop hematologic abnormalities. CH is clearly age dependent and is not rare: at least 10%-20% of people >70 years old carry CH. The newly discovered association between myeloid leukemia-driver mutations and the progression of CVDs has raised medical interest. In this review, we summarize the current view on the contribution of CH in different cardiovascular diseases, CVD risk assessment, patient stratification, and the development of novel therapeutic strategies.

BMP-2 induces human mononuclear cell chemotaxis and adhesion and modulates monocyte-to-macrophage differentiation.

Type 2 diabetes mellitus (T2DM) is a cardiovascular risk factor which leads to atherosclerosis, an inflammatory disease characterized by the infiltration of mononuclear cells in the vessel. Bone morphogenetic protein (BMP)-2 is a cytokine which has been recently shown to be elevated in atherosclerosis and T2DM and to contribute to vascular inflammation. However, the role of BMP-2 in the regulation of mononuclear cell function remains to be established. Herein, we demonstrate that BMP-2 induced human monocyte chemotaxis via phosphoinositide 3 kinase and mitogen-activated protein kinases. Inhibition of endogenous BMP-2 signalling, by Noggin or a BMP receptor inhibitor, interfered with monocyte migration. Although BMP-2 expression was increased in monocytes from T2DM patients, it could still stimulate their migration. Furthermore, BMP-2 interfered with their differentiation into M2 macrophages. Finally, BMP-2 both induced the adhesion of monocytes to fibronectin and endothelial cells (ECs), and promoted the adhesive properties of ECs, by increasing expression of adhesion and pro-inflammatory molecules. Our data demonstrate that BMP-2 could exert its pro-inflammatory effects by inducing monocyte migration and adhesiveness to ECs and by interfering with the monocyte differentiation into M2 macrophages. Our findings provide novel insights into the mechanisms by which BMP-2 may contribute to the development of atherosclerosis.

Low density lipoprotein interferes with intracellular signaling of monocytes resulting in impaired chemotaxis and enhanced chemokinesis.

BACKGROUND: Hypercholesterolemia (HC) is an important cardiovascular risk factor characterized by elevated low density lipoprotein-cholesterol (LDL-C) plasma levels. HC negatively affects monocyte function by reducing their chemotactic response towards different growth factors. We aimed to elucidate the molecular mechanisms by which LDL induces monocyte dysfunction. METHODS AND RESULTS: Human monocytes exposed to either native (nLDL) or oxidized LDL (oxLDL) in vitro showed reduced chemotactic responses towards vascular endothelial growth factor A (VEGFA) and monocyte chemotactic protein-1 (MCP-1), but displayed enhanced random migration (chemokinesis). Mechanistically, the exposure to LDL resulted in the activation of p38 mitogen-activated protein kinase (MAPK) and modulated MCP-1 and VEGFA-induced signaling in human monocytes. Furthermore, the aberrant p38 activation induced by oxLDL is due to the functional impairment of Dual Specificity Phosphatase-1 (DUSP-1). In the absence of LDL, the pharmacological inhibition of DUSP-1 alone was sufficient to recapitulate the accelerated chemokinetic and blunted chemotactic phenotype of monocytes. Finally, p38 MAPK inhibition in monocytes isolated from hyperlipidemic mice prevented the aberrant chemokinetic phenotype. CONCLUSIONS: Our data demonstrate that LDL induces monocyte chemokinesis of human monocytes by inducing mononuclear cell activation through the aberrant modulation of DUSP-1-p38/MAPK signaling axis. Moreover, our findings suggest that MCP-1/VEGFA-induced chemotaxis is reduced by LDL secondary to the impairment of ligand-induced signaling. These findings provide novel insight into hypercholesterolemia-associated vascular dysfunction and its potential involvement in the pathogenesis of atherosclerosis.

TGF-ß-Induced Endothelial-Mesenchymal Transition in Fibrotic Diseases.

Fibrotic diseases are characterized by net accumulation of extracellular matrix proteins in affected organs leading to their dysfunction and ultimate failure. Myofibroblasts have been identified as the cells responsible for the progression of the fibrotic process, and they originate from several sources, including quiescent tissue fibroblasts, circulating CD34⁺ fibrocytes and the phenotypic conversion of various cell types into activated myofibroblasts. Several studies have demonstrated that endothelial cells can transdifferentiate into mesenchymal cells through a process termed endothelial- mesenchymal transition (EndMT) and that this can give rise to activated myofibroblasts involved in the development of fibrotic diseases. Transforming growth factor ß (TGF-ß) has a central role in fibrogenesis by modulating the fibroblast phenotype and function, inducing myofibroblast transdifferentiation and promoting matrix accumulation. In addition, TGF-ß by inducing EndMT may further contribute to the development of fibrosis. Despite extensive investigation of the pathogenesis of fibrotic diseases, no effective treatment strategies are available. Delineation of the mechanisms responsible for initiation and progression of fibrotic diseases is crucial for the development of therapeutic strategies for the treatment of the disease. In this review, we summarize the role of the TGF-ß signaling pathway and EndMT in the development of fibrotic diseases and discuss their therapeutic potential.

Cryopreservation of primary human monocytes does not negatively affect their functionality or their ability to be labelled with radionuclides: basis for molecular imaging and cell therapy.

BACKGROUND: Circulating white blood cells crucially contribute to maintenance and repair of solid organs. Therefore, certain cell populations such as monocytes are attractive targets for use in molecular imaging and cell imaging, e.g. after labelling with radionuclides, as well as for cell therapies. However, the preparation of monocytes may require freezing and thawing to preserve cells for timely and standardised applications. Additional modifications of these cells such as radioisotope labelling are necessary prior to their application in vivo. We therefore tested the hypothesis whether cryopreservation of freshly isolated circulating human monocytes affects their functional phenotype or their suitability for radionuclide labelling. RESULTS: CD14+CD16- monocytes were isolated from human peripheral blood. They were either directly used for cellular assays and labelling or frozen down using cryoprotectants. In the latter case, cells were thawed prior to further use and analysed for survival, chemotactic responses to various growth factors and adhesion on endothelial cells. In addition, both fresh and cryopreserved monocytes were labelled with radiotracers followed by assessment of survival and chemotactic responses. In all functional assays performed, cryopreserved monocytes did not significantly differ from freshly isolated monocytes with regard to their functionality. Cryopreservation did not affect cell survival. There was no effect on the chemotactic response of monocytes towards different growth factors. Likewise, adhesion properties remained unchanged following cryopreservation. Moreover, the labelling efficiency was similar for freshly isolated and cryopreserved monocytes. Labelling did not negatively affect monocyte survival and function. CONCLUSIONS: Our data indicate that cryopreservation of freshly isolated human primary monocytes is feasible and does not negatively affect their functionality when used for labelling and functional assessment.

Activin Receptor-like Kinase 1 Ligand Trap Reduces Microvascular Density and Improves Chemotherapy Efficiency to Various Solid Tumors.

PURPOSE: Antiangiogenic therapy, mostly targeting VEGF, has been applied in cancer patients for the last decade. However, resistance to anti-VEGF therapy and/or no significant benefit as monotherapeutic agent is often observed. Therefore, new antiangiogenic strategies are needed. In the current study, we investigated the therapeutic effect of interfering with the bone morphogenetic protein (BMP)9/activin receptor-like kinase (ALK)1 signaling pathway by using an ALK1-Fc ligand trap. EXPERIMENTAL DESIGN: We analyzed the potential antiangiogenic and antitumor effects of ALK1-Fc protein as monotherapy and in combination with chemotherapy in vivo in mouse models of melanoma, head and neck cancer, and invasive lobular breast carcinomas. ALK1-Fc sequesters BMP9 and 10 and prevents binding of these ligands to endothelial ALK1, which regulates angiogenesis. RESULTS: Treatment of mice with ALK1-Fc strongly decreased the tumors' microvascular density in the three different mouse cancer models. However, this effect was not accompanied by a reduction in tumor volume. An immunohistochemical analysis of the tumor samples revealed that ALK1-Fc treatment increased the pericyte coverage of the remaining tumor vessels and decreased the hypoxia within the tumor. Next, we observed that combining ALK1-Fc with cisplatin inhibited tumor growth in the breast and head and neck cancer models more efficiently than chemotherapy alone. CONCLUSIONS: The addition of ALK1-Fc to the cisplatin treatment was able to enhance the cytotoxic effect of the chemotherapy. Our results provide strong rationale to explore combined targeting of ALK1 with chemotherapy in a clinical setting, especially in the ongoing phase II clinical trials with ALK1-Fc.

ENDOGLIN is dispensable for vasculogenesis, but required for vascular endothelial growth factor-induced angiogenesis.

ENDOGLIN (ENG) is a co-receptor for transforming growth factor-ß (TGF-ß) family members that is highly expressed in endothelial cells and has a critical function in the development of the vascular system. Mutations in Eng are associated with the vascular disease known as hereditary hemorrhagic telangiectasia type l. Using mouse embryonic stem cells we observed that angiogenic factors, including vascular endothelial growth factor (VEGF), induce vasculogenesis in embryoid bodies even when Eng deficient cells or cells depleted of Eng using shRNA are used. However, ENG is required for the stem cell-derived endothelial cells to organize effectively into tubular structures. Consistent with this finding, fetal metatarsals isolated from E17.5 Eng heterozygous mouse embryos showed reduced VEGF-induced vascular network formation. Moreover, shRNA-mediated depletion and pharmacological inhibition of ENG in human umbilical vein cells mitigated VEGF-induced angiogenesis. In summary, we demonstrate that ENG is required for efficient VEGF-induced angiogenesis.

Deficiency for endoglin in tumor vasculature weakens the endothelial barrier to metastatic dissemination.

Therapy-induced resistance remains a significant hurdle to achieve long-lasting responses and cures in cancer patients. We investigated the long-term consequences of genetically impaired angiogenesis by engineering multiple tumor models deprived of endoglin, a co-receptor for TGF-ß in endothelial cells actively engaged in angiogenesis. Tumors from endoglin-deficient mice adapted to the weakened angiogenic response, and refractoriness to diminished endoglin signaling was accompanied by increased metastatic capability. Mechanistic studies in multiple mouse models of cancer revealed that deficiency for endoglin resulted in a tumor vasculature that displayed hallmarks of endothelial-to-mesenchymal transition, a process of previously unknown significance in cancer biology, but shown by us to be associated with a reduced capacity of the vasculature to avert tumor cell intra- and extravasation. Nevertheless, tumors deprived of endoglin exhibited a delayed onset of resistance to anti-VEGF (vascular endothelial growth factor) agents, illustrating the therapeutic utility of combinatorial targeting of multiple angiogenic pathways for the treatment of cancer.

Monocyte function and trafficking in cardiovascular disease.

Monocytes are key effectors of the immune homeostasis and play a crucial role in (vascular) injury repair. Despite their role in immune defense and tissue repair mechanisms, monocytes are also involved in several pathological conditions such as autoimmune and cardiovascular diseases as well as cancer. This suggests that monocytes can be used as diagnostic and as therapeutic targets. A better understanding and characterisation of monocytes and their function in both physiological and pathological situations is thus of great interest. This review focuses on recent advances on the role of monocytes in cardiovascular diseases and describes the value of monocytes as either disease marker or therapeutic target for (cardio)vascular diseases.

BMP signaling in vascular diseases.

Bone morphogenetic proteins (BMPs) are members of the transforming growth factor-ß (TGF-ß) family that signal via type I and type II serine/threonine kinase receptors and intracellular Smad transcription factors. BMPs are multifunctional regulators of development and tissue homeostasis and they were initially characterized as inducers of bone regeneration. Genetic studies in humans and mice showed that perturbations in BMP signaling lead to various diseases, such as skeletal diseases, vascular diseases and cancer. Mutations in BMP type II receptor and BMP type I receptor/activin receptor-like kinase 1 have been linked to pulmonary arterial hypertension and hereditary hemorrhagic telangiectasia, respectively. BMPs have also been implicated in promoting vascular calcification and tumor angiogenesis. In this review we discuss the role of BMP signaling in vascular diseases and the value of BMP signaling as a vascular disease marker or a therapeutic target.

Anti-human activin receptor-like kinase 1 (ALK1) antibody attenuates bone morphogenetic protein 9 (BMP9)-induced ALK1 signaling and interferes with endothelial cell sprouting.

Genetic and molecular studies suggest that activin receptor-like kinase 1 (ALK1), a transforming growth factor ß (TGF-ß) type I receptor, and endoglin, a TGF-ß co-receptor, play an essential role in vascular development and pathological angiogenesis. Several agents that interfere with ALK1 and endoglin function are currently in clinical trials for antiangiogenic activity in cancer therapy. One of these agents, PF-03446962 (anti-hALK1 antibody), shows promising results in the clinic. However, its effects on endothelial cell function and mechanism of action are unclear. Here we demonstrate that anti-hALK1 antibody selectively recognizes human ALK1. The anti-hALK1 antibody interfered with bone morphogenetic protein 9 (BMP9)-induced signaling in endothelial cells. Consistent with this notion, anti-hALK1 antibody was found to compete highly efficiently with the binding of the ALK1 ligand BMP9 and TGF-ß to ALK1. Moreover, it prevented BMP9-dependent recruitment of co-receptor endoglin into this angiogenesis-mediating signaling complex. In addition, we demonstrated that anti-hALK1 antibody inhibited endothelial cell sprouting but did not directly interfere with vascular endothelial growth factor (VEGF) signaling, VEGF-induced proliferation, and migration of endothelial cells. Finally, we demonstrated that BMP9 in serum is essential for endothelial sprouting and that anti-hALK1 antibody inhibits this potently. Our data suggest that both the VEGF/VEGF receptor and the BMP9/ALK1 pathways are essential for stimulating angiogenesis, and targeting both pathways simultaneously may be an attractive strategy to overcome resistance to antiangiogenesis therapy.

Shear induced collateral artery growth modulated by endoglin but not by ALK1.

Transforming growth factor-beta (TGF-ß) stimulates both ischaemia induced angiogenesis and shear stress induced arteriogenesis by signalling through different receptors. How these receptors are involved in both these processes of blood flow recovery is not entirely clear. In this study the role of TGF-ß receptors 1 and endoglin is assessed in neovascularization in mice. Unilateral femoral artery ligation was performed in mice heterozygous for either endoglin or ALK1 and in littermate controls. Compared with littermate controls, blood flow recovery, monitored by laser Doppler perfusion imaging, was significantly hampered by maximal 40% in endoglin heterozygous mice and by maximal 49% in ALK1 heterozygous mice. Collateral artery size was significantly reduced in endoglin heterozygous mice compared with controls but not in ALK1 heterozygous mice. Capillary density in ischaemic calf muscles was unaffected, but capillaries from endoglin and ALK1 heterozygous mice were significantly larger when compared with controls. To provide mechanistic evidence for the differential role of endoglin and ALK1 in shear induced or ischaemia induced neovascularization, murine endothelial cells were exposed to shear stress in vitro. This induced increased levels of endoglin mRNA but not ALK1. In this study it is demonstrated that both endoglin and ALK1 facilitate blood flow recovery. Importantly, endoglin contributes to both shear induced collateral artery growth and to ischaemia induced angiogenesis, whereas ALK1 is only involved in ischaemia induced angiogenesis.

TGFß signaling and cardiovascular diseases.

Transforming growth factor ß (TGFß) family members are involved in a wide range of diverse functions and play key roles in embryogenesis, development and tissue homeostasis. Perturbation of TGFß signaling may lead to vascular and other diseases. In vitro studies have provided evidence that TGFß family members have a wide range of diverse effects on vascular cells, which are highly dependent on cellular context. Consistent with these observations genetic studies in mice and humans showed that TGFß family members have ambiguous effects on the function of the cardiovascular system. In this review we discuss the recent advances on TGFß signaling in (cardio)vascular diseases, and describe the value of TGFß signaling as both a disease marker and therapeutic target for (cardio)vascular diseases.

BMP-7 inhibits TGF-ß-induced invasion of breast cancer cells through inhibition of integrin ß(3) expression.

BACKGROUND: The transforming growth factor (TGF)-ß superfamily comprises cytokines such as TGF-ß and Bone Morphogenetic Proteins (BMPs), which have a critical role in a multitude of biological processes. In breast cancer, high levels of TGF-ß are associated with poor outcome, whereas inhibition of TGF-ß-signaling reduces metastasis. In contrast, BMP-7 inhibits bone metastasis of breast cancer cells. METHODS: In this study, we investigated the effect of BMP-7 on TGF-ß-induced invasion in a 3 dimensional invasion assay. RESULTS: BMP-7 inhibited TGF-ß-induced invasion of the metastatic breast cancer cell line MCF10CA1a, but not of its premalignant precursor MCF10AT in a spheroid invasion model. The inhibitory effect appears to be specific for BMP-7, as its closest homolog, BMP-6, did not alter the invasion of MCF10CA1a spheroids. To elucidate the mechanism by which BMP-7 inhibits TGF-ß-induced invasion, we analyzed invasion-related genes. BMP-7 inhibited TGF-ß-induced expression of integrin α(v)ß(3) in the spheroids. Moreover, targeting of integrins by a chemical inhibitor or knockdown of integrin ß(3) negatively affected TGF-ß-induced invasion. On the other hand, overexpression of integrin ß(3) counteracted the inhibitory effect of BMP7 on TGF-ß-induced invasion. CONCLUSION: Thus, BMP-7 may exert anti-invasive actions by inhibiting TGF-ß-induced expression of integrin ß(3).

TGF-ß1/ALK5-induced monocyte migration involves PI3K and p38 pathways and is not negatively affected by diabetes mellitus.

AIMS: Monocytes contribute to arteriogenesis by infiltration to sites of collateral growth and subsequent production and release of growth factors. Transforming growth factor ß1 (TGF-ß1) mediates monocyte motility and stimulates arteriogenesis. TGF-ß1 signalling mechanisms mediating monocyte motility are unknown so far. Moreover, the influence of cardiovascular risk factor diabetes on TGF-ß1-induced monocyte migration remains to be elucidated. METHODS AND RESULTS: Stimulation of primary human monocytes with TGF-ß1 endorsed phosphorylation of v-Akt murine thymoma viral oncogene analogues protein (AKT), p38, and extracellular signal-related kinase 1/2 (ERK1/2), besides the activation of the SMA/MAD homologues protein (SMAD) pathway. Inhibition of the TGF-ßtype 1 receptor, alias activin receptor-like kinase 5 (ALK5), hindered monocyte chemotaxis towards TGF-ß1 and TGF-ß1-activated downstream signalling cascades. Individual genetic knock-downs for receptor-regulated SMAD2 and SMAD3 did not affect monocyte migration to TGF-ß1. Inhibition of phosphoinositide 3 kinase (PI3K) activity, but not AKT, diminished both basal and TGF-ß1-mediated monocyte motility. TGF-ß1-induced monocyte chemotaxis did not rely on ERK1/2, but rather on p38. Remarkably, TGF-ß1 was able to stimulate chemotaxis of diabetic monocytes. CONCLUSION: The current study provides novel insights into the molecular mechanisms of TGF-ß1-induced monocyte migration, requiring ALK5 kinase activity and signalling via PI3K and p38. TGF-ß1-driven monocyte motogenicity is fully functional in diabetic conditions, which is in sharp contrast to the impaired chemotactic responses to certain other arteriogenic cytokines. Therefore, TGF-ß1 may be a promising candidate for endogenously and exogenously stimulating collateral growth in diabetic patients.

Low oxygen tension positively influences cardiomyocyte progenitor cell function.

Previously we observed that cardiomyocyte progenitor cells (hCMPCs) isolated from the human heart differentiate spontaneously into cardiomyocytes and vascular cells when transplanted after myocardial infarction (MI) in the ischemic heart. After MI, deprivation of oxygen is the first major change in the cardiac environment. How cells handle hypoxia is highly cell type dependent. The effect of hypoxia on cardiac stem or progenitor cells remains to be elucidated. Here, we show for the first time that short- and long-term hypoxia have different effects on hCMPCs. Short-term hypoxia increased the migratory and invasive capacities of hCMPCs likely via mesenchymal transformation. Although long-term exposure to low oxygen levels did not induce differentiation of hCMPCs into mature cardiomyocytes or endothelial cells, it did increase their proliferation, stimulated the secretome of the cells which was shifted to a more anti-inflammatory profile and dampened the migration by altering matrix metalloproteinase (MMP) modulators. Interestingly, hypoxia greatly induced the expression of the extracellular matrix modulator thrombospondin-2 (TSP-2). Knockdown of TSP-2 resulted in increased proliferation, migration and MMP activity. In conclusion, short exposure to hypoxia increases migratory and invasive capacities of hCMPCs and prolonged exposure induces proliferation, an angiogenic secretion profile and dampens migration, likely controlled by TSP-2.