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VEGF-induced intracellular Ca²⁺ oscillations are down-regulated and do not stimulate angiogenesis in breast cancer-derived endothelial colony forming cells.

ABSTRACT: Endothelial colony forming cells (ECFCs) represent a population of truly endothelial precursors that promote the angiogenic switch in solid tumors, such as breast cancer (BC). The intracellular Ca²⁺ toolkit, which drives the pro-angiogenic response to VEGF, is remodelled in tumor-associated ECFCs such that they are seemingly insensitive to this growth factor. This feature could underlie the relative failure of anti-VEGF therapies in cancer patients. Herein, we investigated whether and how VEGF uses Ca²⁺ signalling to control angiogenesis in BC-derived ECFCs (BC-ECFCs). Although VEGFR-2 was normally expressed, VEGF failed to induce proliferation and in vitro tubulogenesis in BC-ECFCs. Likewise, VEGF did not trigger robust Ca²⁺ oscillations in these cells. Similar to normal cells, VEGF-induced intracellular Ca²⁺ oscillations were triggered by inositol-1,4,5-trisphosphate-dependent Ca²⁺ release from the endoplasmic reticulum (ER) and maintained by store-operated Ca²⁺ entry (SOCE). However, InsP₃-dependent Ca²⁺ release was significantly lower in BC-ECFCs due to the down-regulation of ER Ca²⁺ levels, while there was no remarkable difference in the amplitude, pharmacological profile and molecular composition of SOCE. Thus, the attenuation of the pro-angiogenic Ca²⁺ response to VEGF was seemingly due to the reduction in ER Ca²⁺ concentration, which prevents VEGF from triggering robust intracellular Ca²⁺ oscillations. However, the pharmacological inhibition of SOCE prevented BC-ECFC proliferation and in vitro tubulogenesis. These findings demonstrate for the first time that BC-ECFCs are insensitive to VEGF, which might explain at cellular and molecular levels the failure of anti-VEGF therapies in BC patients, and hint at SOCE as a novel molecular target for this disease.

SUBMITTER: Lodola F

PROVIDER: S-EPMC5707017 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Mutations in thin filament regulatory proteins that cause hypertrophic cardiomyopathy (HCM) increase myofilament Ca2+ sensitivity. Mouse models exhibit increased Ca2+ buffering and arrhythmias, and we hypothesized that these changes are primary effects of the mutations (independent of compensatory changes) and that increased Ca2+ buffering and altered Ca2+ handling contribute to HCM pathogenesis via activation of Ca2+-dependent signaling. Here, we determined the primary effects of HCM mutations on intracellular Ca2+ handling and Ca2+-dependent signaling in a model system possessing Ca2+-handling mechanisms and contractile protein isoforms closely mirroring the human environment in the absence of potentially confounding remodeling. Using adenovirus, we expressed HCM-causing variants of human troponin-T, troponin-I, and α-tropomyosin (R92Q, R145G, and D175N, respectively) in isolated guinea pig left ventricular cardiomyocytes. After 48 h, each variant had localized to the I-band and comprised ∼50% of the total protein. HCM mutations significantly lowered the Kd of Ca2+ binding, resulting in higher Ca2+ buffering of mutant cardiomyocytes. We observed increased diastolic [Ca2+] and slowed Ca2+ reuptake, coupled with a significant decrease in basal sarcomere length and slowed relaxation. HCM mutant cells had higher sodium/calcium exchanger activity, sarcoplasmic reticulum Ca2+ load, and sarcoplasmic/endoplasmic reticulum calcium ATPase 2 (SERCA2) activity driven by Ca2+/calmodulin-dependent protein kinase II (CaMKII) phosphorylation of phospholamban. The ryanodine receptor (RyR) leak/load relationship was also increased, driven by CaMKII-mediated RyR phosphorylation. Altered Ca2+ homeostasis also increased signaling via both calcineurin/NFAT and extracellular signal-regulated kinase pathways. Altered myofilament Ca2+ buffering is the primary initiator of signaling cascades, indicating that directly targeting myofilament Ca2+ sensitivity provides an attractive therapeutic approach in HCM.

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VEGF-induced intracellular Ca²⁺ oscillations are down-regulated and do not stimulate angiogenesis in breast cancer-derived endothelial colony forming cells.

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OmicsDI is part of the ELIXIR infrastructure

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Dataset Information

VEGF-induced intracellular Ca2+ oscillations are down-regulated and do not stimulate angiogenesis in breast cancer-derived endothelial colony forming cells.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets

VEGF-induced intracellular Ca²⁺ oscillations are down-regulated and do not stimulate angiogenesis in breast cancer-derived endothelial colony forming cells.