Alkyne has a unique Raman band that does not overlap with Raman scattering from any endogenous molecule in live cells. Here, we show that alkyne-tag Raman imaging (ATRI) is a promising approach for visualizing nonimmobilized small molecules in live cells. An examination of structure-Raman shift/intensity relationships revealed that alkynes conjugated to an aromatic ring and/or to a second alkyne (conjugated diynes) have strong Raman signals in the cellular silent region and can be excellent tags. Using these design guidelines, we synthesized and imaged a series of alkyne-tagged coenzyme Q (CoQ) analogues in live cells. Cellular concentrations of diyne-tagged CoQ analogues could be semiquantitatively estimated. Finally, simultaneous imaging of two small molecules, 5-ethynyl-2'-deoxyuridine (EdU) and a CoQ analogue, with distinct Raman tags was demonstrated.

Click-free imaging of the nuclear localization of an alkyne-tagged cell proliferation probe, EdU, in living cells was achieved for the first time by means of Raman microscopy. The alkyne tag shows an intense Raman band in a cellular Raman-silent region that is free of interference from endogenous molecules. This approach may eliminate the need for click reactions in the detection of alkyne-labeled molecules.

Proliferation, differentiation, and tube formation by endothelial progenitor cells in response to shear stress. Endothelial progenitor cells (EPCs), circulating in peripheral blood, migrate toward target tissue, differentiate, and contribute to the formation of new vessels. In this study, we report that shear stress generated by blood flow or tissue fluid flow can accelerate the proliferation, differentiation, and capillary-like tube formation of EPCs. When EPCs cultured from human peripheral blood were subjected to laminar shear stress, the cells elongated and oriented their long axes in the direction of flow. The cell density of the EPCs exposed to shear stress was higher, and a larger percentage of these cells were in the G 2-M phase of the cell cycle, compared with EPCs cultured under static conditions. Shear stress markedly increased the EPC expression of two vascular endothelial growth factor receptors, kinase insert domain-containing receptor and fms-like tyrosine kinase-1, and an intercellular adhesion molecule, vascular endothelial-cadherin, at both the protein and mRNA levels. Assays for tube formation in the collagen gels showed that the shear-stressed EPCs formed tubelike structures and developed an extensive tubular network significantly faster than the static controls. These findings suggest that EPCs are sensitive to shear stress and that their vasculogenic activities may be modulated by shear stress. blood vessels; angiogenesis; neovascularization; mechanical stress; vascular endothelial growth factor THE FORMATION OF NEW BLOOD vessels in postnatal life has generally been considered to be mediated by the sprouting of endothelial cells (ECs) from preexisting vessels, a process referred to as angiogenesis. However, recent studies have indicated that a vasculogenesis process involving the in situ differentiation of endothelial precursor cells and their subsequent organization into new vessels is also responsible for postnatal neovascularization (10, 22, 27). The existence of bone marrow-derived endothelial progenitor cells (EPCs) circulating in the peripheral blood has been demonstrated in adult humans (6, 26). EPCs have the capacity to circulate, proliferate, and differentiate into mature ECs in response to a variety of growth factors, including VEGF, and other cytokines (7, 16, 18). Transplantation studies have revealed that EPCs can be incorporated into sites of active neovascularization in ischemic hindlimbs and myocardium and contribute to both tumor growth and the formation of new blood vessels (5,20,40). However, the role of EPCs in supporting postnatal vasculogenesis is under intensive investigation, and the factors regulating the migration, proliferation, differentiation, and vessel formation of EPCs are not yet known.During the process of EPC incorporation into tissues and neovascularization, the cells are exposed to fluid shear stress, a mechanical force generated by blood flow or interstitial fluid flow (42). Ample evidence has shown that shear stress modulates mature EC function and gene...

The structure and function of blood vessels adapt to environmental changes such as physical development and exercise. This phenomenon is based on the ability of the endothelial cells to sense and respond to blood flow; however, the underlying mechanisms remain unclear. Here we show that the ATP-gated P2X4 ion channel, expressed on endothelial cells and encoded by P2rx4 in mice, has a key role in the response of endothelial cells to changes in blood flow. P2rx4(-/-) mice do not have normal endothelial cell responses to flow, such as influx of Ca(2+) and subsequent production of the potent vasodilator nitric oxide (NO). Additionally, vessel dilation induced by acute increases in blood flow is markedly suppressed in P2rx4(-/-) mice. Furthermore, P2rx4(-/-) mice have higher blood pressure and excrete smaller amounts of NO products in their urine than do wild-type mice. Moreover, no adaptive vascular remodeling, that is, a decrease in vessel size in response to a chronic decrease in blood flow, was observed in P2rx4(-/-) mice. Thus, endothelial P2X4 channels are crucial to flow-sensitive mechanisms that regulate blood pressure and vascular remodeling.