Towards Novel Geneless Approaches for Therapeutic Angiogenesis

Abstract

Cardiovascular diseases are the leading cause of mortality worldwide. Such a widespread diffusion makes the conditions affecting the heart and blood vessels a primary medical and economic burden. It, therefore, becomes mandatory to identify effective treatments that can alleviate this global problem. Among the different solutions brought to the attention of the medical-scientific community, therapeutic angiogenesis is one of the most promising. However, this approach, which aims to treat cardiovascular diseases by generating new blood vessels in ischemic tissues, has so far led to inadequate results due to several issues. In this perspective, we will discuss cutting-edge approaches and future perspectives to alleviate the potentially lethal impact of cardiovascular diseases. We will focus on the consolidated role of resident endothelial progenitor cells, particularly endothelial colony forming cells, as suitable candidates for cell-based therapy demonstrating the importance of targeting intracellular Ca²⁺ signaling to boost their regenerative outcome. Moreover, we will elucidate the advantages of physical stimuli over traditional approaches. In particular, we will critically discuss recent results obtained by using optical stimulation, as a novel strategy to drive endothelial colony forming cells fate and its potential in the treatment of cardiovascular diseases.

Keywords: cardiovascular disease; cell fate; conjugated polymers; endothelial colony forming cells; intracellular Ca2+ signaling; optical stimulation; therapeutic angiogenesis; transient receptor potential vanilloid 1.

Figure 1

Endothelial colony forming cells (ECFCs) Ca²⁺ machinery and pro-angiogenic Ca²⁺ signals. Growth factors (like VEGF or IGF2) and chemokines (like SDF-1) bind to Receptor tyrosine kinases (RTKs) and G protein-coupled receptors (GPCRs) respectively, thus activating specific PLC isoforms, which in turn leads to production of inositol 1,4,5-trisphosphate (InsP₃). InsP₃ binds to InsP₃ receptors (InsP₃R) bearing the release of Ca²⁺ from the endoplasmic reticulum (ER) pool. The Ca²⁺ store depletion, detected by Ca²⁺ sensor Stromal interaction molecule 1 (STIM1), is the signal for store-operated calcium entry (SOCE) activation. SOCE, the major Ca²⁺ entry pathway in ECFCs, is mediated by the interaction among STIM1 and the proteins Orai1 and Transient receptor potential canonical 1 (TRPC1). These plasma membrane pore channels allow Ca²⁺ entry from the extracellular space that will be subsequently transported from the cytosol within the SR by the Sarco-Endoplasmic Reticulum Calcium ATPase (SERCA), while Plasma membrane Calcium ATPase (PMCA) contributes to clear cytosolic Ca²⁺ levels. Transient receptor potential vanilloid (TRPV) channels (TRPV1 and TRPV4) also represent an alternative pathway for extracellular Ca²⁺ entry in ECFCs. These intracellular Ca²⁺ signals evoke pathways (NF-κB, ERK1/2, PI3K/Akt) that lead to nuclear transcription of pro-angiogenic factors.

Figure 2

Conjugated polymers optically drive the fate of Endothelial Colony Forming Cells. (A) Sketch of the polymer device used for cell optical activation. ECFCs are cultured on top of P3HT thin films, deposited on glass substrates. Optical excitation is provided by a green LED (λ_MAX^EM 525 nm). (B) Photo-thermal and photo-electrochemical reactions occur at the interface between material and ECFC membrane. The latter is the predominant mechanism triggering TRPV1 activation. A subsequent increase in [Ca²⁺]_i results in the degradation of IκB, the inhibitory sub-unit of the transcriptional factor NF-κB. As a consequence, the p65 NF-κB subunit is released from IκB inhibition and translocates into the nucleus leading to a robust up-regulation of angiogenic genes, which are under NF-κB-dependent transcriptional control.