The dynamic regulation of blood vessel caliber☆☆☆★★★

Background 

The flow of blood to organs is regulated by changes in the diameter of the blood vessels. Because the flow through a vessel is directly related to the radius3, small changes in the diameter can have marked changes in flow.1 The diameter of the blood vessels is dependent on a dynamic interaction between two cell types: the endothelial cells and the smooth muscle cells. The endothelium is a fragile monolayer of cells on the inner surface of the vessel. The endothelial cells are exposed to a variety of events, including the mechanical forces of pressure and flow (shear stress), drugs, hormones, and toxins (Fig 1).

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Fig. 1. The blood vessel. Endothelial cells (green ovals) are exposed to a variety of stimuli, including mechanical forces of pressure and flow (shear stress), drugs, hormones, and toxins. Endothelial cells transduce these stimuli and respond by sending messages to smooth muscle cells (red ovals) , which leads to responses such as contraction, relaxation, proliferation, migration, or synthesis of proteins.

Our understanding of endothelial cell function, and a core principle of vascular biology, is based on a relatively simple observation. Furchgott and Zawadzki2 showed that strips of blood vessels, in which the endothelial layer was present, relaxed in response to acetylcholine. However, if the endothelial layer were removed, the blood vessels then contracted in response to acetylcholine. They took their observations one step further and placed a vessel with an intact endothelium next to a vessel without endothelium. In this case, when acetylcholine was added, the vessel without endothelium then relaxed in response to acetylcholine. On the basis of these experiments, they proposed that the stimulation of the endothelium with acetylcholine led to the production of a soluble substance that caused muscle relaxation. They named this substance endothelium-derived relaxing factor. It took another 7 years to identify this substance as the simple gas nitric oxide (NO).1 Since then, it has been shown that the endothelium can produce a variety of other substances, which in turn effect smooth muscle responses.

Physiology of contraction 

Although the endothelium has an important role in the transduction of stimuli from the blood interface, it is the underlying smooth muscle cells that perform the work of maintaining tone. The smooth muscle in the media of the vessel controls the dynamic caliber of the vessel by the state of contraction or relaxation of the muscle cells. Our understanding of the mechanisms of contraction in well-ordered muscle cells, such as cardiac or skeletal muscle, is clearer than our understanding of smooth muscle. The smooth muscle is a syncytium of spindle-shaped cells. Although the internal structure of smooth muscle is not completely defined, it has best been described as containing four separate domains. The first is the contractile domain, which contains the thick filaments (myosin), the thin filaments (α-actin), and the thin filament regulatory proteins (calponin, caldesmon, tropomyosin, and small heat shock proteins). The second domain is the cytoskeletal domain, which contains β–non-muscle actin and the intermediate filaments (desmin and vimentin). The contractile and cytoskeletal domains are connected at two other domains: the focal contact points on the membrane, dense plaques, and the focal contact points inside the cell, dense bodies. Thus, these focal contact points may serve as regulatory centers where the contractile and cytoskeletal domains converge.

Our understanding of vascular smooth muscle physiology begins with the events that occur when a contractile agonist, such as norepinephrine, interacts with receptors on the membrane of the cell. This initiates a cascade of signals, the first of which is an increase in the calcium concentration inside the cells. This increase in calcium activates a kinase (myosin light chain kinase) that phosphorylates a regulatory subunit on the myosin motor protein (the regulatory myosin light chain), leading to a conformational change in the head of the myosin molecule and the consumption of energy. This results in the formation and cycling of cross-bridges between actin and myosin, the shortening of the muscle, and the generation of force (Fig 2).

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Fig. 2. Myosin (yellow) is a motor protein that, when activated, leads to formation of cross-bridges with actin and sliding of actin thin filaments to a more shortened configuration (thick filament regulation). Actin (thin yellow line) is associated with a variety of proteins, including tropomyosin, caldesmon, calponin, and small heat shock proteins, which may be important in maintenance of force (thin filament regulation). We propose a model for maintenance of force in smooth muscle (tethering domain model) in which phosphorylated HSP27 (P-HSP27; orange oval) tethers actin with a specific but unknown domain protein (green squares) . During relaxation, phosphorylated HSP20 (P-HSP20, purple triangle) directly interacts with phosphorylated HSP27, thus destabilizing interaction of actin with domain protein.

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Fig. 3. Thick filament regulation of smooth muscle contraction. Increases in calcium (Ca 2+) activate myosin light chain kinase (MLCK), which phosphorylates regulatory light chains (MLC) on the myosin motor molecule. Regulatory light chains are dephosphorylated by myosin light chain phosphatase (MLC Ppase) , which has recently been shown to be inhibited by Rho-activated kinase (ROK) . Thick filament regulation of smooth muscle contraction assumes that force is dependent on the phosphorylation state of regulatory myosin light chains.

Unlike cardiac or skeletal muscles, vascular smooth muscle is able to maintain force for long periods of time. One of the confounding questions in smooth muscle physiology is that this sustained phase of muscle contraction is associated with calcium concentrations, myosin light chain phosphorylation, and levels of energy consumption that are near those found in muscles that are not contracted (Fig 4).

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Fig. 4. Relationship between calcium concentrations, myosin light chain phosphorylation, and force development in vascular smooth muscle contraction. Stimulation of vascular smooth muscles with a contractile agonist (A) leads to rapid increases in calcium concentrations (dotted line) followed by increases in phosphorylation of regulatory myosin light chains (dashed lines) and development of force (solid line) . During the sustained phase of muscle contraction, calcium concentrations and myosin light chain phosphorylation return to near basal levels.

Physiology of relaxation 

There are two ways in which contracted muscles decrease force or relax. The first involves the removal of a contractile agonist (passive relaxation). Alternatively, in the continued presence of a contractile agonist, substances that activate other signaling pathways, such as NO and prostacyclin, and that will cause relaxation (active relaxation). NO leads to increases in cyclic guanosine monophosphate (cGMP), whereas prostacyclin leads to increases in cyclic adenosine monophosphate (cAMP). cGMP and cAMP then activate cGMP-dependent protein kinase and cAMP-dependent protein kinase, respectively. Kinases are enzymes that phosphorylate specific substrate proteins, and multiple substrate proteins have been implicated in muscle relaxation (Table I).

Table I. Mechanisms of cyclic nucleotide-dependent relaxation

	Substrate	Mechanism of action
	Kca channels	Repolarize muscle membrane
	L-type Ca2+ channels	Promote release of Ca2+
	IP3 receptor, phospholamban	Inhibits release of Ca2+ from intracellular stores
	Myosin light chain kinase	Inhibits myosin light chain phosphorylation
	MLC20 phosphatase	Dephosphorylates MLC20
	HSP20	Dissociates relaxation and the Ca2+-dependent MLC20 pathway

Possible cyclic nucleotide-dependent protein kinase substrates in smooth muscle include: plasma membrane ion channels (Kca or L-type Ca2+ channels; IP3; phospholamban, which activates the adenosine triphosphatase of the endoplasmic reticulum, leading to a decrease in intracellular Ca2+); myosin light chain kinase, which reduces its affinity for Ca2+, resulting in decreased phosphorylation of the MLC20; or the MLC20 phosphatase, which dephophorylates MLC20. These putative cyclic nucleotide-dependent protein kinase substrates all mediate smooth muscle relaxation through the decrease of either intracellular calcium concentrations or MLC20 phosphorylation. However, increases in the phosphorylation of the small heat shock–related protein, HSP20, may dissociate relaxation from the Ca2+ dependent MLC20 pathway. K Ca, Calcium-dependent potassium channel; Ca 2+, calcium; IP 3, inositol 1,4,5-triphosphate receptor; MLC 20, myosin light chains; HSP20, heat shock protein 20.

Our laboratory has recently shown that one of the proteins that is phosphorylated during active relaxation is a small heat shock–related protein, HSP20.4 More direct evidence that suggests that this protein is important in the relaxation of muscle is that phosphorylated peptide analogues of HSP20 inhibit muscle contraction.5 Although the specific functions of heat shock proteins in muscle cells are not known, heat shock proteins often function as “molecular chaperones” and directly modulate protein interactions. There are considerable amounts of two of the small heat shock proteins, HSP20 and HSP27, in muscle cells, and both of these proteins are associated with actin. Thus, the small heat shock proteins may have an important role in the regulation of smooth muscle physiology.

Proposed model 

On the basis of this new information, we propose a model for the maintenance of force in vascular smooth muscle: the tethering domain model (Fig 2). This model suggests that the small heat shock proteins stabilize and destabilize force at a specific domain in the muscle cell. It is possible that HSP27 stabilizes actin with a specific, but yet unknown, structural element in the muscle cell. One candidate for this structural element is the dense bodies and dense plaques that are strategically located where the structural rearrangements that are required for shortening of muscle cells may occur. Because the activation of relaxation pathways leads to increases in the phosphorylation of HSP20, the phosphorylated HSP20 might directly interact with HSP27, leading to a conformational change in the HSP27 molecule. This would result in a dissociation of HSP27 and actin from the specific tethering attachment and a relaxation of the muscle cells. An alternate, but also untested, model to explain the dynamic cellular rearrangements that occur with muscle contraction and relaxation is that there are changes in actin filament dynamics (actin polymerization and depolymerization). Because both HSP27 and HSP20 modulate actin filament dynamics, this model is also plausible.

Blood vessel tone and vascular remodeling 

Two processes have been implicated in the chronic changes in vessel diameter that occur with atherosclerosis and restenosis after angioplasty: intimal thickening and remodeling. Smooth muscle cell migration, proliferation, and the production of matrix molecules have been implicated in intimal thickening. The activation of cyclic nucleotide-dependent signaling pathways has been shown to inhibit vascular smooth muscle proliferation and migration in vitro and in vivo. Remodeling refers to the changes in artery wall structure in response to hemodynamic forces. Remodeling maintains shear stress and wall tension within a physiologic range. There is growing evidence that it is the failure of normal remodeling, rather than the accumulation of intimal plaque, that leads to both atherosclerosis and restenosis after angioplasty. The specific mechanisms that account for physiologic and pathologic remodeling are not known. However, it is likely that endothelial-derived NO and the impact of NO on the smooth muscle is important in this process. Thus, the same signaling events that are activated during smooth muscle relaxation may be important in the modulation of chronic changes in vessel caliber.

Future questions 

The possibility that stress proteins regulate smooth muscle physiology suggests molecular mechanisms for the association between stress and vascular diseases, such as hypertension and atherosclerosis. Future studies are needed to determine whether there is a relationship between the processes that regulate acute changes in vessel caliber with the processes that regulate chronic changes in caliber (remodeling). In addition, studies are needed to determine the mechanisms by which the heat shock proteins modulate vasomotor tone. The answer to these questions will lead to a better understanding of vascular physiology and to more direct approaches to the treatment of vascular diseases.