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

Decomposition cross-correlation for analysis of collagen matrix deformation by single smooth muscle cells

Microvascular remodeling is known to depend on cellular interactions with matrix tissue. However, it is difficult to study the role of specific cells or matrix elements in an in vivo setting. The aim of this study is to develop an automated technique that can be employed to obtain and analyze local collagen matrix remodeling by single smooth muscle cells. We combined a motorized microscopic setup and time-lapse video microscopy with a new cross-correlation based image analysis algorithm to enable automated recording of cell-induced matrix reorganization. This method rendered 60–90 single cell studies per experiment, for which collagen deformation over time could be automatically derived. Thus, the current setup offers a tool to systematically study different components active in matrix remodeling

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

Serotonylation of Vascular Proteins Important to Contraction

BACKGROUND:Serotonin (5-hydroxytryptamine, 5-HT) was named for its source (sero-) and ability to modify smooth muscle tone (tonin). The biological effects of 5-HT are believed to be carried out by stimulation of serotonin receptors at the plasma membrane. Serotonin has recently been shown to be synthesized in vascular smooth muscle and taken up from external sources, placing 5-HT inside the cell. The enzyme transglutaminase uses primary amines such as 5-HT to covalently modify proteins on glutamine residues. We tested the hypothesis that 5-HT is a substrate for transglutaminase in arterial vascular smooth muscle, with protein serotonylation having physiological function. METHODOLOGY/PRINCIPAL FINDINGS:The model was the rat aorta and cultured aortic smooth muscle cells. Western analysis demonstrated that transglutaminase II was present in vascular tissue, and transglutaminase activity was observed as a cystamine-inhibitable incorporation of the free amine pentylamine-biotin into arterial proteins. Serotonin-biotin was incorporated into alpha-actin, beta-actin, gamma-actin, myosin heavy chain and filamin A as shown through tandem mass spectrometry. Using antibodies directed against biotin or 5-HT, immunoprecipitation and immunocytochemistry confirmed serotonylation of smooth muscle alpha-actin. Importantly, the alpha-actin-dependent process of arterial isometric contraction to 5-HT was reduced by cystamine. CONCLUSIONS:5-HT covalently modifies proteins integral to contractility and the cytoskeleton. These findings suggest new mechanisms of action for 5-HT in vascular smooth muscle and consideration for intracellular effects of primary amines

Flow inhibits inward remodeling in cannulated porcine small coronary arteries

Flow inhibits inward remodeling in cannulated porcine small coronary arteries. Am J Physiol Heart Circ Physiol 289: H2632-H2640, 2005. First published July 15, 2005; doi: 10.1152/ajpheart. 00205.2005. -The mechanisms of flow-induced vascular remodeling are poorly understood, especially in the coronary microcirculation. We hypothesized that application of flow in small coronary arteries in organoid culture would cause a nitric oxide (NO)-mediated dilation and inhibit inward remodeling. We developed an organoid culture setup to drive a flow through cannulated arterioles at constant luminal pressure via a pressure gradient between the pipettes. Subepicardial porcine coronary arterioles with diameter at full dilation and 60 mmHg (D-0) of 168 +/- 10 (SE) mu m were cannulated. Vessels treated with N omega-nitro-L-arginine (L-NNA) to block NO production and untreated vessels were pressurized at 60 mmHg for 3 days with and without flow. Endothelium-dependent dilation to 10(-7) M bradykinin was preserved in all groups. Tone was significantly less in vessels cultured under flow conditions in the last half of the culture period. Untreated and L-NNA-treated vessels regulated their diameter to yield shear stresses of 10.3 +/- 2.1 and 14.0 +/- 2.4 (SE) dyn/cm(2), respectively (not significantly different). Without L-NNA, passive pressure-diameter curves at the end of the culture period revealed inward remodeling in the control group [ to 92.3 +/- 1.3% of D-0 (SE)] and no remodeling in the vessels cultured under flow conditions (100.2 +/- 1.3% of D-0); with L-NNA, the group subjected to flow showed inward remodeling (92.1 +/- 2.5% of D-0). We conclude that pressurized coronary resistance arteries could be maintained in culture for several days with flow. Vessels cultured under flow conditions remained more dilated when NO synthesis was blocked. Inward remodeling occurred in vessels cultured under no-flow conditions and was inhibited by flow-dependent NO synthesi

Mechanics of microvascular remodeling

This paper reviews work on microvascular remodeling that has been done over the past years in our lab. It is not our purpose to fully cover the field; rather we explain our progress in a more or less chronological order. We address physiological and pathological remodeling in resistance vessels, the biomechanics of the vascular wall and the factors that determine vascular caliber. Subsequently, the intimate link between maintained vascular tone and inward remodeling is discussed, and we highlight our view that tone and remodeling form hallmarks in a continuous process of vascular adaptation. Finally, the role of transglutaminases in remodeling is describe