RESUMO
Transient receptor potential vanilloid 1 (TRPV1) is a nonselective cation channel that is important for nociception and inflammatory pain and is activated by a variety of nociceptive stimuliâincluding lipids such as capsaicin (CAP) and endocannabinoids. TRPV1's role in physiological systems is often studied by activating it with externally perfused ligands; however, this approach is plagued by poor spatiotemporal resolution. Lipid agonists are insoluble in physiological buffers and can permeate membranes to accumulate nonselectively inside cells, where they can have off-target effects. To increase the spatiotemporal precision with which we can activate lipids on cells and tissues, we previously developed optically cleavable targeted (OCT) ligands, which use protein tags (SNAP-tags) to localize a photocaged ligand on a target cellular membrane. After enrichment, the active ligand is released on a flash of light to activate nearby receptors. In our previous work, we developed an OCT-ligand to control a cannabinoid-sensitive GPCR. Here, we expand the scope of OCT-ligand technology to target TRPV1 ion channels. We synthesize a probe, OCT-CAP, that tethers to membrane-bound SNAP-tags and releases a TRPV1 agonist when triggered by UV-A irradiation. Using Ca2+ imaging and electrophysiology in HEK293T cells expressing TRPV1, we demonstrate that OCT-CAP uncaging activates TRPV1 with superior spatiotemporal precision when compared to standard diffusible ligands or photocages. This study is the first example of an OCT-ligand designed to manipulate an ion-channel target. We anticipate that these tools will find many applications in controlling lipid signaling pathways in various cells and tissues.
RESUMO
While hyaluronic acid (HA)-based hydrogels have been used clinically for decades, the mechanisms by which HA exerts molecular weight-dependent bioactivity and how chemical modification and crosslinking may affect molecular weight-dependent bioactivity remain poorly understood. This knowledge gap presents a significant barrier to designing HA hydrogels with predictable bioactivities. As HA has been widely reported to have molecular weight-dependent effects on endothelial cells (ECs), we investigated how the molecular weight of HA in either soluble or crosslinked forms affects angiogenesis and interrogated CD44 clustering on the surface of endothelial cells as a candidate mechanism for these affects. Using soluble HA, our results show high molecular weight (HMW) HA, but not low molecular weight (LMW) HA, increased viability and tube formation in cultured human cerebral microvascular ECs (HCMVECs). No size of HA affected proliferation. When HCMVECs were cultured with crosslinked HA of varying molecular weights in the form of HA-based microporous annealed particle scaffold (HMAPS), the cell response was comparable to when cultured with soluble HA. Similarly, when implanted subcutaneously, HMAPS with HMW HA were more vascularized than those with LMW HA. We also show that antibody-mediated CD44 clustering resulted in HCMVECs with increased viability and tube-like structure formation in a manner comparable to exposure to HMW HA, suggesting that HMW acts through CD44 clustering. STATEMENT OF SIGNIFICANCE: Biomaterials based on hyaluronic acid (HA), a bioactive extracellular matrix polysaccharide, have been used in clinical products for several years. Despite the knowledge that HA molecular weight heavily influences its bioactivity, molecular weight has been largely ignored in the development of HA-based biomaterials. Given the high viscosity of high molecular weight HA typically found in native tissues, lower molecular weight polysaccharides have been used most commonly for biomaterial fabrication. By comparing the ability of injectable, microporous annealed particle scaffolds (MAPS) fabricated from variably sized HA to promote angiogenesis, this study demonstrates that MAPS with high molecular weight HA better support vascularization, likely through an unique ability to induce clustering of CD44 receptors on endothelial cells.