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Optogenetics provides promising tools for the precise control of receptor-mediated cell behaviors in a spatiotemporal manner. Yet, most photoreceptors require extensive genetic manipulation and respond only to ultraviolet or visible light, which are suboptimal for in vivo applications because they do not penetrate thick tissues. Here we report a novel near-infrared light-activated DNA agonist (NIR-DA) nanodevice for nongenetic manipulation of cell signaling and phenotype in deep tissues. This nanodevice is prepared by conjugating a preinactivated DNA agonist onto the gold nanorods (AuNRs). Upon NIR light treatment, the DNA agonist is released through the localized surface plasmon resonance (LSPR)-based photothermal effect of AuNRs and becomes active. The active DNA agonist dimerizes the DNA-modified chimeric or native receptor tyrosine kinase (RTK) on cell surfaces and activates downstream signal transduction in live cells. Such NIR-DA activation of RTK signaling enables the control of cytoskeletal remodeling, cell polarization, and directional migration. Furthermore, we demonstrate that the NIR-DA system can be used in vivo to mediate RTK signaling and skeletal muscle satellite cell migration and myogenesis, which are critical cellular behaviors in the process of skeletal muscle regeneration. Thus, the NIR-DA system offers a powerful and versatile platform for exogenous modulation of deep tissues for purposes such as regenerative medicine.

Bioinspired molecular engineering strategies have emerged as powerful tools that significantly enhance the development of novel therapeutics, improving efficacy, specificity, and safety in disease treatment. Recent advancements have focused on identifying and utilizing disease‐associated biomarkers to optimize drug activity and address challenges inherent in traditional therapeutics, such as frequent drug administrations, poor patient adherence, and increased risk of adverse effects. In this review, we provide a comprehensive overview of the latest developments in bioinspired artificial systems (BAS) that use molecular engineering to tailor therapeutic responses to drugs in the presence of disease‐specific biomarkers. We examine the transition from open‐loop systems, which rely on external cues, to closed‐loop feedback systems capable of autonomous self‐regulation in response to disease‐associated biomarkers. We detail various BAS modalities designed to achieve biomarker‐driven therapy, including activatable prodrug molecules, smart drug delivery platforms, autonomous artificial cells, and synthetic receptor‐based cell therapies, elucidating their operational principles and practical in vivo applications. Finally, we discuss the current challenges and future perspectives in the advancement of BAS‐enabled technology and envision that ongoing advancements toward more programmable and customizable BAS‐based therapeutics will significantly enhance precision medicine. [ABSTRACT FROM AUTHOR]

The actin-myosin interaction of vascular smooth muscle cells (VSMCs) is regulated by myosin light chain kinase (MLCK), which is a fusion protein of the central catalytic domain with the N-terminal actin-binding and C-terminal myosin-binding domains. In addition to the regulatory role of kinase activity mediated by the catalytic domain, nonkinase activity that derives from both terminals is able to exert a regulatory role as reviewed by Nakamura et al. (32). We previously showed that nonkinase activity mediated the filopodia upon the stimulation by sphingosylphosphorylcholine (SPC) (25). To explore the regulatory role of nonkinase activity in chemotaxis, we constructed VSMCs where the expression of MLCK was totally abolished by using a lentivirus-mediated RNAi system. We hypothesized that the MLCK-downregulated VSMCs were unable to form filopodia and to migrate upon SPC stimulation and confirmed the hypothesis. We further constructed a kinase-inactive mutant from bovine cDNA coding wild-type (WT) MLCK by mutating the ATP-binding sites located in the catalytic domain, followed by confirming the presence (absence) of the kinase activity of WT (kinase-inactive mutant). We transfected WT and the mutant into MLCK-downregulated VSMCs. We expected that the transfected VSMCs will recover the ability to induce filopodia and chemotaxis toward SPC and found both constructs rescued the ability. Because they share the actin- and myosin-binding domains, we concluded nonkinase activity plays a major role for SPC-induced migration. chemotaxis; myosin light chain kinase

Blebbistatin is a myosin II-specific inhibitor. However, the mechanism and tissue specificity of the drug are not well understood. Blebbistatin blocked the chemotaxis of vascular smooth muscle cells (VSMCs) toward sphingosylphosphorylcholine (I[C.sub.50] = 26.1 [+ or -] 0.2 and 27.5 [+ or -] 0.5 [micro]M for GbaSM-4 and A7r5 cells, respectively) and platelet-derived growth factor BB (I[C.sub.50] = 32.3 [+ or -] 0.9 and 31.6 [+ or -] 1.3 [micro]M for GbaSM-4 and A7r5 cells, respectively) at similar concentrations. Immunofluorescence and fluorescent resonance energy transfer analysis indicated a blebbistatin-induced disruption of the actin-myosin interaction in VSMCs. Subsequent experiments indicated that blebbistatin inhibited the [Mg.sup.2+]-ATPase activity of the unphosphorylated (I[C.sub.50] = 12.6 [+ or -] 1.6 and 4.3 [+ or -] 0.5 [micro]M for gizzard and bovine stomach, respectively) and phosphorylated (I[C.sub.50] = 15.0 [+ or -] 0.6 [micro]M for gizzard) forms of purified smooth muscle myosin II, suggesting a direct effect on myosin II motor activity. It was further observed that the [Mg.sup.2+]-ATPase activities of gizzard myosin II fragments, heavy meromyosin (I[C.sub.50] = 14.4 [+ or -] 1.6 [micro]M) and subfragment 1 (I[C.sub.50] = 5.5 [+ or -] 0.4 [micro]M), were also inhibited by blebbistatin. Assay by in vitro motility indicated that the inhibitory effect of blebbistatin was reversible. Electron-microscopic evaluation showed that blebbistatin induced a distinct conformational change (i.e., swelling) of the myosin II head. The results suggest that the site of blebbistatin action is within the S1 portion of smooth muscle myosin II. Boyden chamber; fluorescence resonance energy transfer; ATPase; in vitro motility assay; electron microscopy

Molecular mechanisms underlying migration of vascular smooth muscle cells (VSMCs) toward sphingosylphosphorylcholine (SPC) were analyzed in light of the hypothesis that remodeling of the actin cytoskeleton should be involved. After SPC stimulation, mitogen-activated protein kinases (MAPKs), including p38 MAPK (p38) and p42/44 MAPK (p42/44), were found to be phosphorylated. Migration of cells toward SPC was reduced in the presence of SB-203580, an inhibitor of p38, but not PD-98059, an inhibitor of p42/44. Pertussis toxin (PTX), a [G.sub.i] protein inhibitor, induced an inhibitory effect on p38 phosphorylation and VSMC migration. Myosin light chain (MLC) phosphorylation occurred after SPC stimulation with or without pretreatment with SB-203580 or PTX. The MLC kinase inhibitor ML-7 and the Rho kinase inhibitor Y-27632 inhibited MLC phosphorylation but only partially inhibited SPC-directed migration. Complete inhibition was achieved with the addition of SB-203580. After SPC stimulation, the actin cytoskeleton formed thick bundles of actin filaments around the periphery of cells, and the cells were surrounded by elongated filopodia, i.e., magunapodia. The peripheral actin bundles consisted of [alpha]- and [beta]-actin, but magunapodia consisted exclusively of [beta]-actin. Such a remodeling of actin was reversed by addition of SB-203580 and PTX, but not ML-7 or Y-27632. Taken together, our biochemical and morphological data confirmed the regulation of actin remodeling and suggest that VSMCs migrate toward SPC, not only by an MLC phosphorylation-dependent pathway, but also by an MLC phosphorylation-independent pathway. arteriosclerosis; cytoskeletal remodeling doi:10.1152/ajpheart.00901.2005