Vascular smooth muscle cells remodel collagen matrices by long-distance action and anisotropic interaction

While matrix remodeling plays a key role in vascular physiology and pathology, the underlying mechanisms have remained incompletely understood. We studied the remodeling of collagen matrices by individual vascular smooth muscle cells (SMCs), clusters and monolayers. In addition, we focused on the contribution of transglutaminase 2 (TG2), which plays an important role in the remodeling of small arteries. Single SMCs displaced fibers in collagen matrices at distances up to at least 300 μm in the course of 8–12 h. This process involved both ‘hauling up’ of matrix by the cells and local matrix compaction at a distance from the cells, up to 200 μm. This exceeded the distance over which cellular protrusions were active, implicating the involvement of secreted enzymes such as TG2. SMC isolated from TG2 KO mice still showed compaction, with changed dynamics and relaxation. The TG active site inhibitor L682777 blocked local compaction by wild type cells, strongly reducing the displacement of matrix towards the cells. At increasing cell density, cells cooperated to establish compaction. In a ring-shaped collagen matrix, this resulted in preferential displacement in the radial direction, perpendicular to the cellular long axis. This process was unaffected by inhibition of TG2 cross-linking. These results show that SMCs are capable of matrix remodeling by prolonged, gradual compaction along their short axis. This process could add to the 3D organization and remodeling of blood vessels based on the orientation and contraction of SMCs

A comparison of responses to raised extracellular potassium and endothelium-derived hyperpolarizing factor (EDHF) in rat pressurised mesenteric arteries

The present study examined the hypothesis that potassium ions act as an endothelium-derived hyperpolarizing factor (EDHF) released in response to ACh in small mesenteric arteries displaying myogenic tone. Small mesenteric arteries isolated from rats were set up in a pressure myograph at either 60 or 90 mmHg. After developing myogenic tone, responses to raising extracellular potassium were compared to those obtained with ACh (in the presence of nitric oxide synthase and cyclo- oxygenase inhibitors). The effects of barium and ouabain, or capsaicin, on responses to raised extracellular potassium or ACh were also determined. The effects of raised extracellular potassium levels and ACh on membrane potential, were measured using sharp microelectrodes in pressurised arteries. Rat small mesenteric arteries developed myogenic tone when pressurised. On the background of vascular tone set by a physiological stimulus (i.e pressure), ACh fully dilated the small arteries in a concentration-dependent manner. This response was relatively insensitive to the combination of barium and ouabain, and insensitive to capsaicin. Raising extracellular potassium produced a more inconsistent and modest vasodilator response in pressurised small mesenteric arteries. Responses to raising extracellular potassium were sensitive to capsaicin, and the combination of barium and ouabain. ACh caused a substantial hyperpolarisation in pressurized arteries, while raising extracellular potassium did not. These data indicate that K+ is not the EDHF released in response to ACh in myogenically active rat mesenteric small arteries. Since the hyperpolarization produced by ACh was sensitive to carbenoxolone, gap junctions are the likely mediator of EDH responses under physiological conditions

The Redox State of Transglutaminase 2 Controls Arterial Remodeling

While inward remodeling of small arteries in response to low blood flow, hypertension, and chronic vasoconstriction depends on type 2 transglutaminase (TG2), the mechanisms of action have remained unresolved. We studied the regulation of TG2 activity, its (sub) cellular localization, substrates, and its specific mode of action during small artery inward remodeling. We found that inward remodeling of isolated mouse mesenteric arteries by exogenous TG2 required the presence of a reducing agent. The effect of TG2 depended on its cross-linking activity, as indicated by the lack of effect of mutant TG2. The cell-permeable reducing agent DTT, but not the cell-impermeable reducing agent TCEP, induced translocation of endogenous TG2 and high membrane-bound transglutaminase activity. This coincided with inward remodeling, characterized by a stiffening of the artery. The remodeling could be inhibited by a TG2 inhibitor and by the nitric oxide donor, SNAP. Using a pull-down assay and mass spectrometry, 21 proteins were identified as TG2 cross-linking substrates, including fibronectin, collagen and nidogen. Inward remodeling induced by low blood flow was associated with the upregulation of several anti-oxidant proteins, notably glutathione-S-transferase, and selenoprotein P. In conclusion, these results show that a reduced state induces smooth muscle membrane-bound TG2 activity. Inward remodeling results from the cross-linking of vicinal matrix proteins, causing a stiffening of the arterial wall

Thrombospondin-1 in Early Flow-Related Remodeling of Mesenteric Arteries from Young Normotensive and Spontaneously Hypertensive Rats

We tested the hypotheses that TSP-1 participates in the initiation of remodeling of small muscular arteries in response to altered blood flow and that the N-terminal domain of TSP-1 (hepI) can reverse the pathological inward remodeling of resistance arteries from SHR