**5. Blood flow, vascular hemodynamics and neural control**

The cardiovascular system is a closed circuit comprised of the heart and an elaborate network of vessels in the systemic and pulmonary circulations. Biophysical forces and factors that govern transportation of essential gases and metabolic fuels and nutrients as well as waste products of metabolism are termed "hemodynamics" and are largely determined by local tissue and cellular supply and demand balance. Hemodynamics, especially in the systemic circulation, are regulated by intrinsic (ability of local tissues to regulate their blood supply) and extrinsic (reliance on neural mechanisms to distribute blood flow at the tissue level) neural control systems. These complex regulatory mechanisms rely on the ability of blood vessels to properly respond to chemical cues in order to efficiently orchestrate where and how much blood is being delivered and removed from various vascular beds. Dysfunctional intrinsic and extrinsic neural control systems compromise efficient oxygen and nutrient delivery to tissues and manifests in a variety of disease pathologies such as PAD, stroke, and/or heart attack.

As oxygenated blood leaves the left ventricle of the heart, it enters the aortic arch and exerts biophysical forces on the arterial walls. Driving pressure, transmural pressure, and hydrostatic pressure are all important forces that regulate blood flow and two major components of these forces are tensile stress and shear stress. Tensile stress is the perpendicular force exerted by flowing blood on the vessel wall and represents forces due to blood pressure [3]. In comparison, fluid shear stress is the force parallel or tangential to the vessel wall which corresponds to the frictional force of the blood in contact with the intimal endothelium [4]. These combined forces are critical for stimulating both intrinsic and extrinsic neural control mechanisms in the vasculature.

#### **5.1. Autonomic nervous system and the vasculature**

States and worldwide for many years [1,2]. Despite major advances in our knowledge of the numerous contributing mechanisms for CVD and potential therapeutic strategies against CVD, estimates suggest CVD-related deaths will continue to rise over the next 20 years or so [1,2]. Certainly, notwithstanding these significant basic science and clinical advances, failure in adequate clinical control of CVD highlights its complexity and points to the need for continued study of the underlying mechanisms of and potential routes for control of CVD.

**Figure 2. Vascular smooth muscle cell function.** VSM cells are normally quiescent and operate via vasoconstriction/ vasorelaxation to control local blood flows and pressures and downstream tissue perfusion. However, VSM dysfunc‐ tion leads to dedifferentiation to an embryonic phenotype, and under these pathologic conditions VSM loses its con‐ tractile characteristics and becomes synthetic, proliferative, and migratory, serving as basis for many forms of CVD. Also shown in this schematic are cellular features of thick and thin filaments, dense plaques, and the cytoskeleton.

**-Contractile -Quiescent**

**FIGURE 2**

8 Muscle Cell and Tissue

**-Vasoconstriction/vasodilation**

**-Synthetic -Proliferative -Migratory**

Of the many forms of CVD perhaps the most common is an occlusive disorder termed atherosclerosis. Atherosclerosis is a gradual and progressive disease that involves combined influences of heightened inflammatory status, locally dysfunctional metabolism, abnormal vascular wall growth and remodeling, and the buildup of an occlusive plaque. As discussed, the involvement of uncontrolled VSM growth is a basic foundation of the evolution phase of atherosclerosis, and dedifferentiation of VSM cells into a growth-promoting and synthetic phenotype contributes largely to the emerging and growing plaque. This process of enhanced synthesis and proliferation of medial VSM cells can be slow and progressive and can occur over many decades and may not elicit observable symptoms. This pathology often eventuates in a stenotic plaque, either stable or complicated, which can then become jeopardized and lead to lumen obstruction and diminution of blood flow with clear repercussions of reduced oxygen and nutrient delivery to downstream tissues and diminished removal of toxic metabolites and gases. The plaque can also rupture and send microthrombi into the downstream circulation which can lodge in smaller blood vessels. If atherosclerosis occurs in the peripheral circulation, then these processes could manifest as tissue necrosis with perhaps loss of the affected tissue or limb. Of heightened significance, if this pathology occurs in the cerebral or myocardial circulation, this could result in a cerebral or myocardial infarct (stroke or heart attack) with

critical and life-threatening implications.

The autonomic nervous system is comprised of the complementary yet distinct sympathetic (SNS) and parasympathetic (PSNS) nervous systems. Under normal physiological conditions, these systems perform divergent regulatory functions on the vasculature. In general, the SNS typically acts to constrict blood vessels, effectively reducing blood flow, while the PSNS generally dilates blood vessels to increase flow. Autonomic dysfunction in the context of abnormally high blood pressure is typically the result of an overly active SNS. This dysfunction produces a constant neuronal discharge of sympathetic neurons resulting in tonic vasocon‐ striction of the VSM [5].

The SNS contains preganglionic sympathetic neurons in the ventral horn of the thoracolumbar spinal cord which project their axons just outside of the sympathetic trunk and synapse with postganglionic sympathetic neurons near its target organ. At the initial synapse, depolariza‐ tion of the presynaptic neuron triggers tethering, docking, and fusion of vesicles containing the neurotransmitter Acetylcholine (Ach) to the axon hillock. Once Ach is released from the presynaptic neuron, it binds to an ionotropic, nicotinic, cholinergic receptor on the postgan‐ glionic neuron, resulting in depolarization on the postsynaptic cell. In the context of carotid artery blood flow, the postganglionic neuron projects onto, and synapses with, medial VSM cells. At the postganglionic synapse, the neurotransmitter that is released is primarily norepi‐ nephrine (NE). In similar fashion, vesicles containing NE are released at the postganglionic synapse, yet they bind to metabotropic, adrenergic receptors on the VSM cells. VSM contains both alpha 1 and beta 2 adrenergic receptors. NE will preferentially bind to the excitatory alpha 1 receptors causing vasoconstriction and reduced blood flow. Since it is well documented that the carotid artery is tonically constricted, it is only after inhibition of this signal that the SNS is able to be turned off by the PSNS, thereby resulting in carotid artery dilation and enhanced cerebral blood flow. Essentially, the vasomotor area of the medulla is sending excitatory, efferent output to the thoracolumbar section of the spinal cord which is innervating and contracting carotid VSM. This action is executed until the vasomotor area receives an inhibitory signal from the Nucleus Tractus Solitarii (NTS), at which point the SNS is inhibited while the PSNS is activated.

In comparison to the constitutively active SNS, the PSNS is alternatively activated by the carotid sinus when the carotid artery pressure increases. This means that as luminal pressure exerts increased force on the vessel wall, the carotid sinus increases its firing frequency and amplitude, activating the PSNS and effectively inhibiting SNS tonic discharge. The purpose of its activation is to dilate the artery in an attempt to normalize or depress the increased pressure that is currently acting on the vessel walls. The PSNS projects its preganglionic neurons directly from the brainstem and/or sacral spinal cord towards, and in close proximity to, its target organ(s). At this synapse, the preganglionic neuron releases Ach in a similar fashion as that described for the SNS. The ensuing depolarization of the postganglionic neuron reaches the carotid artery and releases Ach again, yet the mechanisms of action at this second synapse are different than what occurs at the initial synapse. Here, Ach binds to beta 2, metabotropic, muscarinic receptors located on the endothelium adjacent to the VSM cells in the medial layer. After binding to the appropriate G-protein coupled receptor on the endothelium, a signaling cascade is initiated that results in an influx of intracellular calcium and the synthesis of nitric oxide (NO) from endothelial nitric oxide synthase (eNOS). NO can then stimulate broad and multifaceted actions on downstream processes including those associated with cyclic nucleo‐ tide signaling (discussed below).

#### **5.2. Intrinsic neural control mechanisms**

Arterial tensile stress elicits myogenic tone and primarily modulates mechanoreceptor feedback found in baroreceptors within conduit arteries (discussed below). Myogenic tone is the inherent and tonic contractile response of VSM to increased luminal pressures and is a critical component for determining vessel wall hypertrophy according to LaPlace's equation: T = r x P (T = tension; r = vessel radius; and P = pressure), whereby if vessel caliber or diameter remains constant, any increase in intravascular pressure will elicit an increase in wall tension. Since these two factors are proportional, the vessel wall thickens in order to help offset any experienced increase in wall tension. In the high pressure environment of the systemic vasculature failure to adapt can result in increased matrix metalloproteinase (MMP) activity, causing vessel wall thinning and ensuing aneurysmal formation [6]. The other key force in regulating blood flow is fluid shear stress. This tangential force is exerted on intimal endothe‐ lium and elicits VSM relaxation through canonical dilatory (largely NO-mediated) pathways. The mechanical stimulation of intimal VECs by luminal blood flow activates nonselective cation channels (TRP channels) within the cellular plasma membrane [7]. The resulting increase in intracellular calcium facilitates activation of eNOS and yields NO as a by-product of L-arginine to L-citrulline conversion [8]. As a diatomic gas, NO freely diffuses across the VEC membrane and travels in paracrine fashion to abluminal VSM cells. Here, NO binds to the heme moiety on soluble guanylate cyclase (sGC) and dephosphorylates guanosine triphophosphate (GTP) to elicit pyrophosphate (PPi) and cyclic GMP. Two cyclic GMP molecules can then bind to the regulatory subunit of the serine/threonine (Ser/Thr) protein kinase G (PKG), which in turn phosphorylates a vast number of intracellular proteins to include ion channels, phospholamban, myosin light chain phosphatase, and other phosphor‐ ylatable targets including vasodilator-stimulated serum phosphoprotein (VASP), a topic of interest discussed later in this chapter. Ultimately, PKG activation results in the resequestration of calcium within the sarcoplasmic reticulum and induces VSM relaxation resulting in vessel dilation and increased blood flow.

#### **5.3. Extrinsic neural control mechanisms**

nephrine (NE). In similar fashion, vesicles containing NE are released at the postganglionic synapse, yet they bind to metabotropic, adrenergic receptors on the VSM cells. VSM contains both alpha 1 and beta 2 adrenergic receptors. NE will preferentially bind to the excitatory alpha 1 receptors causing vasoconstriction and reduced blood flow. Since it is well documented that the carotid artery is tonically constricted, it is only after inhibition of this signal that the SNS is able to be turned off by the PSNS, thereby resulting in carotid artery dilation and enhanced cerebral blood flow. Essentially, the vasomotor area of the medulla is sending excitatory, efferent output to the thoracolumbar section of the spinal cord which is innervating and contracting carotid VSM. This action is executed until the vasomotor area receives an inhibitory signal from the Nucleus Tractus Solitarii (NTS), at which point the SNS is inhibited while the

In comparison to the constitutively active SNS, the PSNS is alternatively activated by the carotid sinus when the carotid artery pressure increases. This means that as luminal pressure exerts increased force on the vessel wall, the carotid sinus increases its firing frequency and amplitude, activating the PSNS and effectively inhibiting SNS tonic discharge. The purpose of its activation is to dilate the artery in an attempt to normalize or depress the increased pressure that is currently acting on the vessel walls. The PSNS projects its preganglionic neurons directly from the brainstem and/or sacral spinal cord towards, and in close proximity to, its target organ(s). At this synapse, the preganglionic neuron releases Ach in a similar fashion as that described for the SNS. The ensuing depolarization of the postganglionic neuron reaches the carotid artery and releases Ach again, yet the mechanisms of action at this second synapse are different than what occurs at the initial synapse. Here, Ach binds to beta 2, metabotropic, muscarinic receptors located on the endothelium adjacent to the VSM cells in the medial layer. After binding to the appropriate G-protein coupled receptor on the endothelium, a signaling cascade is initiated that results in an influx of intracellular calcium and the synthesis of nitric oxide (NO) from endothelial nitric oxide synthase (eNOS). NO can then stimulate broad and multifaceted actions on downstream processes including those associated with cyclic nucleo‐

Arterial tensile stress elicits myogenic tone and primarily modulates mechanoreceptor feedback found in baroreceptors within conduit arteries (discussed below). Myogenic tone is the inherent and tonic contractile response of VSM to increased luminal pressures and is a critical component for determining vessel wall hypertrophy according to LaPlace's equation: T = r x P (T = tension; r = vessel radius; and P = pressure), whereby if vessel caliber or diameter remains constant, any increase in intravascular pressure will elicit an increase in wall tension. Since these two factors are proportional, the vessel wall thickens in order to help offset any experienced increase in wall tension. In the high pressure environment of the systemic vasculature failure to adapt can result in increased matrix metalloproteinase (MMP) activity, causing vessel wall thinning and ensuing aneurysmal formation [6]. The other key force in regulating blood flow is fluid shear stress. This tangential force is exerted on intimal endothe‐ lium and elicits VSM relaxation through canonical dilatory (largely NO-mediated) pathways.

PSNS is activated.

10 Muscle Cell and Tissue

tide signaling (discussed below).

**5.2. Intrinsic neural control mechanisms**

Of all our organs that receive systemic blood flow, the cerebral circulation is of critical importance. According to the American Heart Association, stroke is a leading cause of death and the leading cause of adult disability in the United States [1]. Ischemic stroke, hemorrhagic stroke, and transient ischemic attacks all result from failure of oxygenated blood to reach the brain. In order to maintain appropriate cerebral blood pressure and constant brain perfusion, numerous cardiovascular and neural control mechanisms exist that act cooperatively to ensure adequate and appropriate cerebral circulation. The carotid sinus baroreceptor reflex is partly responsible for maintaining and regulating blood flow to the brain through local and systemic regulation of blood pressures. The carotid sinus innervates the internal carotid artery just distal to the bifurcation of the common carotid artery. Here, the sensory fibers of the carotid sinus extend into the medial VSM layer of the internal carotid artery. These sensory nerve endings sense stretch (via geometric alteration of resident TRP channels) within the blood vessel and follow the baroreceptor reflex arc. The source of this stretch is typically caused by an increase in intraluminal pressure, which forces the vessel to expand in diameter in order to contain an elevated blood volume within a defined enclosed space (the vessel walls). These volume and pressure changes are also reflected as increased transmural pressure that is exerted by the freely flowing blood on the vessel wall and incidentally on the nerve terminals of the carotid sinus. Following stimulation, an excitatory signal is generated by an influx of cations entering the nerve terminal and the generation of a depolarization event. This afferent signal travels up the sinus nerve and connects to the glossopharyngeal nerve, which proceeds and terminates at the NTS in the brainstem. Once the signal has reached the brainstem, a series of excitatory or inhibitory interneurons communicate and are either stimulated or inhibited, which will differentially activate or inhibit one of the two divisions of the autonomic nervous system previously described. However, under abnormal conditions in the cerebral vasculature or carotid arteries, these control mechanisms may become compromised, thereby resulting in dysfunctional baroreceptor reflex mechanisms and compromised cerebral blood flow [5,9]. On a broader scale, any obstruction or dysfunction at any point along this reflex arc may be instrumental in the pathogenesis of stroke and/or CVD as a whole [10].

#### **5.4. Physiological blood flow profiles**

Four types of physiological blood flow dominate the circulation: 1) pulsatile, 2) oscillatory, 3) laminar, and 4) turbulent [4,11]. Pulsatile and oscillatory blood flows are similar and result from periodic fluctuations in the upstream pumping of the heart in relation to downstream "vacuum" or pulling forces generated by the respiratory and skeletal muscles as well as changing metabolic demands. Since balance between laminar and turbulent flow is important for moderation of flow-mediated stresses and the development of CVD, ensuing discussion focuses on these two forms of blood flow. Continuous (unidirectional) laminar blood flow represents uninterrupted flow that occurs at or near the capillary level and is frequently studied in cell culture perfusion systems or computer modeling simulations. Laminar flow is characterized by layered blood flow in the absence of detectable blood velocity fluctuations or turbulence (e.g., eddies, whorls, and flow reversals). By convention, blood flow is laminar when Reynold's number (Re), a ratio of inertial to viscous forces of a solution, is below 2000, where Re=2rvp/n with r = radius, v = velocity, p = density, and n = viscosity. Re exceeds 3000 under conditions of turbulent flow. When Re is high, it means that the inertial forces outweigh the viscous forces of the liquid. Because stenotic vessels create a pressure drop distal to the site of occlusion, blood velocity increases at these sites and creates audible turbulence with extremely high Re values. Turbulent blood flow, then, is associated with changes in the layered context of flow as seen in laminar settings and is correlated with increased stresses on the vessel wall. Turbulent blood flow is also caused by branching or arborization of blood vessels (at bifurcations of arteries, for example) or by lesions or plaque located along the luminal wall which creates flow obstructions. Any local vascular region with a calculated Re > 3000 is associated with elevated risk for developing vascular occlusions (plaque) at that site.

Considering the importance of laminar flow in maintaining proper vascular homeostasis and turbulent or erratic blood flow in contributing to vascular dysfunction underlying CVD, we began studies aimed at investigating the influence of altered blood flow and flow-mediated shear stress on VSM migration. Using a newly developed in vitro flow apparatus with controlled fluid viscosity (ViscoLab4000) and a newly developed in vitro wound healing and migration assay (Ibidi microchannel VI.4), preexposure of rat VSM cells to 4 hours of elevated shear stress (10 dyn/cm2 ) significantly reduced serum-stimulated migration compared to static controls (SC) with no observable changes in cell morphology through 16 hours (Figure 3). These early findings support our notion that elevated fluid shear stress protects against enhanced VSM cell migration as occurs in the setting of CVD.

These results demonstrate the importance of homeostatic levels of fluid shear in maintaining normal vessel function and are commensurate with other studies that have shown this time point and level of fluid shear stress to be physiologically relevant in reducing migration using non-in-situ migration assays [12,13]. It is important to accept the findings of these previous studies with some prudence since removing cells from their perfusion location and seeding them elsewhere can disrupt focal adhesions and other cytoskeletal arrangements formed in

**FIGURE 3**

a broader scale, any obstruction or dysfunction at any point along this reflex arc may be

Four types of physiological blood flow dominate the circulation: 1) pulsatile, 2) oscillatory, 3) laminar, and 4) turbulent [4,11]. Pulsatile and oscillatory blood flows are similar and result from periodic fluctuations in the upstream pumping of the heart in relation to downstream "vacuum" or pulling forces generated by the respiratory and skeletal muscles as well as changing metabolic demands. Since balance between laminar and turbulent flow is important for moderation of flow-mediated stresses and the development of CVD, ensuing discussion focuses on these two forms of blood flow. Continuous (unidirectional) laminar blood flow represents uninterrupted flow that occurs at or near the capillary level and is frequently studied in cell culture perfusion systems or computer modeling simulations. Laminar flow is characterized by layered blood flow in the absence of detectable blood velocity fluctuations or turbulence (e.g., eddies, whorls, and flow reversals). By convention, blood flow is laminar when Reynold's number (Re), a ratio of inertial to viscous forces of a solution, is below 2000, where Re=2rvp/n with r = radius, v = velocity, p = density, and n = viscosity. Re exceeds 3000 under conditions of turbulent flow. When Re is high, it means that the inertial forces outweigh the viscous forces of the liquid. Because stenotic vessels create a pressure drop distal to the site of occlusion, blood velocity increases at these sites and creates audible turbulence with extremely high Re values. Turbulent blood flow, then, is associated with changes in the layered context of flow as seen in laminar settings and is correlated with increased stresses on the vessel wall. Turbulent blood flow is also caused by branching or arborization of blood vessels (at bifurcations of arteries, for example) or by lesions or plaque located along the luminal wall which creates flow obstructions. Any local vascular region with a calculated Re > 3000 is

associated with elevated risk for developing vascular occlusions (plaque) at that site.

Considering the importance of laminar flow in maintaining proper vascular homeostasis and turbulent or erratic blood flow in contributing to vascular dysfunction underlying CVD, we began studies aimed at investigating the influence of altered blood flow and flow-mediated shear stress on VSM migration. Using a newly developed in vitro flow apparatus with controlled fluid viscosity (ViscoLab4000) and a newly developed in vitro wound healing and migration assay (Ibidi microchannel VI.4), preexposure of rat VSM cells to 4 hours of elevated

controls (SC) with no observable changes in cell morphology through 16 hours (Figure 3). These early findings support our notion that elevated fluid shear stress protects against

These results demonstrate the importance of homeostatic levels of fluid shear in maintaining normal vessel function and are commensurate with other studies that have shown this time point and level of fluid shear stress to be physiologically relevant in reducing migration using non-in-situ migration assays [12,13]. It is important to accept the findings of these previous studies with some prudence since removing cells from their perfusion location and seeding them elsewhere can disrupt focal adhesions and other cytoskeletal arrangements formed in

enhanced VSM cell migration as occurs in the setting of CVD.

) significantly reduced serum-stimulated migration compared to static

instrumental in the pathogenesis of stroke and/or CVD as a whole [10].

**5.4. Physiological blood flow profiles**

12 Muscle Cell and Tissue

shear stress (10 dyn/cm2

**Figure 3. Fluid shear stress reduces migration of VSM cells.** Using a newly developed laser capture microdissection (LCM)-assisted wounding assay to estimate cell migration, rat primary VSM cells exposed to increased shear stress show significantly reduced ability to migrate in response to serum compared to static control (SC) cells. Photomicro‐ graphs show confluent VSM cells exposed to a wounding scrape injury and treated with static flow (SC) or elevated flow (shear stress 10 dyn/cm2 ) for 4 hours with continual exposure through 16 hours. **A** and **B** are photos taken after 4 hours pretreatment of 0 (SC) or 10 dyn/cm2 , and **C** and **D** represent photos taken after continual exposure for 16 hours. Arrows shown in **B** and **D** represent direction of flow. **E** shows measurements of percent recovery of the wound width normalized to time 0 for SC and flow-exposed cells.

response to fluid flow and that are necessary for proper cellular and cytoskeletal dynamics. Due to this shortcoming, we developed this novel wound-healing assay that utilizes an ultraviolet laser and LCM microscopy (Zeiss Palm Laser LCM) to precisely control and effectively denude adherent VSM cells from the perfusion channel substrate following exposure to fluid shear stress in a more physiological setting (without the aforementioned adverse confounding effects found in many traditional migration assays). This new technique allows the researcher to perfuse and injure cells in the same location without the deleterious effects of trypsinization and disruption of normal cellular architecture (including, notably, focal adhesions and cytoskeletal components). Channels are promptly washed with complete media prior to imaging at time zero. Migration time points are carried out up to 16 hours in order to prevent proliferation effects on wound closure rates. To further increase the accuracy and throughput of our migration measurements, we will use the motorized stage of the confocal microscope that is equipped with X, Y, Z coordinate control and on-stage incubator to perform simultaneous time-lapse analysis on multiple wounds.

As mentioned, one of the main functions of a healthy endothelial layer is to provide a key interface between blood flowing in the lumen and the underlying subintimal layer. When this functional endothelium becomes jeopardized, increased fluid shear stress experienced by the underlying medial VSM cells negatively contributes to arterial dysfunction. It has been shown that increased continuous laminar flow can improve re-endothelialization and attenuate VSM cell migration; however, in certain pathologies increased pulsatile and turbulent flow sensed by exposed VSM and the presence of physical obstructions such as intimal plaque promotes VSM cell proliferation and migration as underpinnings of deleterious vessel growth and remodeling [14,15]. This pathogenic feed-forward mechanism has poor implications for proper vessel function and tissue perfusion.
