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Acute mechanoadaptation of vascular smooth muscle cells in response to continuous arteriolar vasoconstriction: implications for functional remodeling ¹

LUIS A. MARTINEZ-LEMUS^*, MICHAEL A. HILL, STEFFEN S. BOLZ, ULRICH POHL and GERALD A. MEININGER^*,2

^* Department of Medical Physiology, Cardiovascular Research Institute-Division of Vascular Biology, Texas A&M University System Health Science Center, College Station, Texas, USA;
Microvascular Biology Group, School of Medical Sciences, RMIT University, Bundoora, Victoria, Australia, and
Physiology Institute, Ludwig Maximilians University, Munich, Germany

²Correspondence: Department of Medical Physiology, Reynolds Medical Bldg., Room 349, Texas A&M University System, Health Science Center, College Station, TX 77843-1114, USA. E-mail: gam{at}tamu.edu

SPECIFIC AIMS

Arteriolar vasoconstriction, sustained for as little as 4 h, has been associated with an acute functional remodeling phenomenon characterized by a delayed and incomplete relaxation of the arterioles upon removal of the vasoconstrictor stimulus. As more prolonged vasoconstrictor stimuli have been associated with remodeling of the vascular wall cellular components, we performed a series of experiments aimed at determining whether vascular smooth muscle cells (VSMC) undergo a change in cell shape or positional rearrangement within the vascular wall as a form of mechanoadaptation, in response to prolonged vasoconstriction. This was accomplished by characterizing changes in the 3-dimensional position and morphology of VSMC in isolated skeletal muscle arterioles during short (5 min) and prolonged (4 h) norepinephrine (NE)-induced vasoconstriction.

PRINCIPAL FINDINGS

1. During acute initial vasoconstriction (5 min NE exposure), VSMC within the wall of arterioles display varying degrees of shortening as vessel diameter decreases in response to NE such that there is a corresponding degree of cells sliding relative to neighboring cells
To visualize VSMC within the wall of living isolated arterioles, we developed a novel fluorescent dye exclusion labeling technique. The technique is based on the principle that carboxyfluorescein added abluminally into the bathing solution of isolated, cannulated, and pressurized arterioles is excluded from living cells so that it produces a shadow image of individual VSMC within the vascular wall without the need for fixation. Through-focus optical image slices were obtained by multiphoton microscopy and reconstructed into 3-dimensional sets to provide visualization of individual VSMC over time. To characterize the morphological changes VSMC undergo upon rapid vasoconstriction, isolated skeletal muscle arterioles were mounted into a pressure myograph and constricted for 5 min with NE (10^–5.5 M) while under observation using the exclusion labeling technique. All VSMC exhibited a reduction in length 5 min after exposure to NE, but the degree of shortening observed was variable (5–44%). Due to the VSMC heterogeneity in shortening, reduction in mean VSMC length was smaller (76.3±3.8% of control, n=7) than the mean reduction in vascular diameter (61.3±2.6% of control, n=7), which in conjunction with an increased number of cell cross sections observed at a mid-diameter view of the vessel indicated the cells were able to slide relative to each other during the initial constriction. This was corroborated by the observation that, upon constriction, VSMC wrapped around a greater proportion of the vessel circumference than before vasoconstriction (Fig. 1 ). Upon removal of NE after the 5 min exposure, VSMC returned to their original length and position within the vascular wall.

View larger version (33K): [in this window] [in a new window]   Figure 1. Proportion of vessel circumference occupied by vascular smooth muscle cell length and expressed as percent of control, before, during, and after short (5 min, n=7) and long (4 h, n=13) constriction to 10–5.5 mol/L norepinephrine (NE). *P < 0.05 vs control conditions.

2. During prolonged exposure to NE (4 h), the initial VSMC shortening is followed by cellular elongation in the continued presence of vasoconstriction. The cellular elongation suggests that VSMC undergo a mechanoadaptation process (length autoregulation) as a mechanism for efficiently maintaining a vasoconstricted state
When NE exposure and constriction were extended to 4 h, VSMC within the arteriolar wall returned toward their original length. This was evidenced as an increase in cell length and cell movement relative to neighboring cells during the maintained vasoconstriction: 56% of the cells returned to their original length. This autoregulation of length resulted in cells returning to between 50 and 102% of their control length (41.4±14.1 average % compensation.) The tapered ends of adjacent VSMC were observed to increase their degree of overlap by 6.86 ± 1.8 µm (Fig. 2 ). These observations indicate that in the prolonged presence of a vasoconstrictor stimulus, VSMC undergo a mechanoadaptation process after cell shortening that involves cellular elongation, allowing cells to return toward their original length (length autoregulation) while maintaining a reduced diameter.

View larger version (52K): [in this window] [in a new window]   Figure 2. 3-Dimensional reconstruction (frontal view) of a rat cremaster arteriole after both 5 min and 4 h of exposure to 10–5.5 mol/L norepinephrine (NE). Two tapered vascular smooth muscle cell ends were digitally colored to illustrate the increase in cell overlap occurring during the 4 h exposure to NE while the vessel maintained its reduced diameter.

3. In response to 4 h of continuous NE-induced vasoconstriction, arterioles fail to return to their control diameters after removal of NE whereas VSMC return to their control cell lengths
At the end of the prolonged 4 h NE-induced vasoconstriction, vessels were washed with fresh physiological saline solution without NE for as long as 30 min. After removal of the NE for 30 min, vessel diameters did not return to control values. Vessels remained partially constricted (74±2.8% of control, n=13). At the same time, average VSMC lengths did return to their original control values. The proportion of the vessel circumference occupied by the VSMC remained increased (139±7.7% of control, n=13.) These observations suggest that the impaired relaxation after long-term NE exposure may result from a functional repositioning or remodeling of VSMC within the vascular wall such that the cells at their original length constrain the vessel wall so that diameter is reduced.

CONCLUSIONS

In summary, the impaired relaxation after prolonged NE exposure, as we have reported, is independent of a sustained increase in vascular smooth muscle calcium and dependent on tyrosine phosphorylation. We have defined this impairment as resulting from a functional remodeling of the arteriolar wall. We report here that the impaired relaxation is associated with a unique mechanoadaptation process involving mechanisms for autoregulation of VSMC length. After acute vasoconstriction, cellular shortening leads to a reduction in vessel diameter. However, if the vessel remains constricted for a prolonged period, then a population of VSMC within the vessel wall adapt by a process involving cellular elongation. This occurs despite a maintained vasoconstriction and without causing vascular dilation. We hypothesize that the observed VSMC mechanoadaptation underlies functional remodeling. It is speculated that this functional remodeling process may be important for long-term autoregulation of tissue blood flow and may represent an initial step in eutrophic remodeling. We propose that this mechanoadaptive process acts to maintain a reduced diameter without the need for cells to maintain a shortened state through continued actin–myosin interaction or latch. This would be an economical cellular mechanism (energetically) to preserve a reduced diameter for prolonged periods.

View larger version (52K): [in this window] [in a new window]   Figure 3. Schematic representation of the mechanoadaptive process in which vascular smooth muscle cells (VSMC) undergo cellular elongation after initial shortening (i.e., length autoregulation) in response to maintained (4 h) exposure to the vasoconstrictor norepinephrine (NE). A) Representation of an arteriole under control conditions with spontaneous myogenic tone. B) Representation of an arteriole 5 min after the initial exposure to NE. Due to the heterogeneity in constriction level among the VSMC, some degree of cell slippage occurred, resulting in an increase of the proportion one VSMC wraps around the vessel circumference. C) Representation of an arteriole after 4 h of continuous exposure to NE. The vessel remains constricted with a diameter similar to that observed at the initial 5 min exposure to NE. During the 4 h exposure to NE and despite the presence of a sustained constriction, a population consisting of 56% of the VSMC (represented by the single cells on the right side panel) has been returning toward original length increasing the degree of overlap between adjacent cells (represented by the red and green cells within the vessel wall.) D) Representation of an arteriole 30 min after the removal of NE. Although vessel diameter remains partially constricted, average VSMC length is back to control values. We propose that VSMC that began returning toward control length during the sustained constriction are forming new attachments with neighboring cells and extracellular matrices such that when NE is removed, they return to original length before the vessel diameter reaches its pre-NE value. Thus, it is the new cellular attachments from this population of cells that prevent the vessel from returning to its original diameter and underlie the observed functional remodeling.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.03-0634fje; doi: 10.1096/fj.03-0634fje

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