Patrick W. Alford

Biohybrid thin films for measuring contractility in engineered cardiovascular muscle

In vitro cardiovascular disease models need to recapitulate tissue-scale function in order to provide in vivo relevance. We have developed a new method for measuring the contractility of engineered cardiovascular smooth and striated muscle in vitro during electrical and pharmacological stimulation. We present a growth theory-based finite elasticity analysis for calculating the contractile stresses of a 2D anisotropic muscle tissue cultured on a flexible synthetic polymer thin film. Cardiac muscle engineered with neonatal rat ventricular myocytes and paced at 0.5 Hz generated stresses of 9.2 +/- 3.5 kPa at peak systole, similar to measurements of the contractility of papillary muscle from adult rats. Vascular tissue engineered with human umbilical arterial smooth muscle cells maintained a basal contractile tone of 13.1 +/- 2.1 kPa and generated another 5.1 +/- 0.8 kPa when stimulated with endothelin-1. These data suggest that this method may be useful in assessing the efficacy and safety of pharmacological agents on cardiovascular tissue.

Cyclic strain induces dual-mode endothelial-mesenchymal transformation of the cardiac valve

Endothelial-mesenchymal transformation (EMT) is a critical event for the embryonic morphogenesis of cardiac valves. Inducers of EMT during valvulogenesis include VEGF, TGF-β1, and wnt/β-catenin (where wnt refers to the wingless-type mammary tumor virus integration site family of proteins), that are regulated in a spatiotemporal manner. EMT has also been observed in diseased, strain-overloaded valve leaflets, suggesting a regulatory role for mechanical strain. Although the preponderance of studies have focused on the role of soluble mitogens, we asked if the valve tissue microenvironment contributed to EMT. To recapitulate these microenvironments in a controlled, in vitro environment, we engineered 2D valve endothelium from sheep valve endothelial cells, using microcontact printing to mimic the regions of isotropy and anisotropy of the leaflet, and applied cyclic mechanical strain in an attempt to induce EMT. We measured EMT in response to both low (10%) and high strain (20%), where low-strain EMT occurred via increased TGF-β1 signaling and high strain via increased wnt/β-catenin signaling, suggesting dual strain-dependent routes to distinguish EMT in healthy versus diseased valve tissue. The effect was also directionally dependent, where cyclic strain applied orthogonal to axis of the engineered valve endothelium alignment resulted in severe disruption of cell microarchitecture and greater EMT. Once transformed, these tissues exhibited increased contractility in the presence of endothelin-1 and larger basal mechanical tone in a unique assay developed to measure the contractile tone of the engineered valve tissues. This finding is important, because it implies that the functional properties of the valve are sensitive to EMT. Our results suggest that cyclic mechanical strain regulates EMT in a strain magnitude and directionally dependent manner.

Blast-induced phenotypic switching in cerebral vasospasm

Vasospasm of the cerebrovasculature is a common manifestation of blast-induced traumatic brain injury (bTBI) reported among combat casualties in the conflicts in Afghanistan and Iraq. Cerebral vasospasm occurs more frequently, and with earlier onset, in bTBI patients than in patients with other TBI injury modes, such as blunt force trauma. Though vasospasm is usually associated with the presence of subarachnoid hemorrhage (SAH), SAH is not required for vasospasm in bTBI, which suggests that the unique mechanics of blast injury could potentiate vasospasm onset, accounting for the increased incidence. Here, using theoretical and in vitro models, we show that a single rapid mechanical insult can induce vascular hypercontractility and remodeling, indicative of vasospasm initiation. We employed high-velocity stretching of engineered arterial lamellae to simulate the mechanical forces of a blast pulse on the vasculature. An hour after a simulated blast, injured tissues displayed altered intracellular calcium dynamics leading to hypersensitivity to contractile stimulus with endothelin-1. One day after simulated blast, tissues exhibited blast force dependent prolonged hypercontraction and vascular smooth muscle phenotype switching, indicative of remodeling. These results suggest that an acute, blast-like injury is sufficient to induce a hypercontraction-induced genetic switch that potentiates vascular remodeling, and cerebral vasospasm, in bTBI patients.

A Possible Role for Integrin Signaling in Diffuse Axonal Injury

Over the past decade, investigators have attempted to establish the pathophysiological mechanisms by which non-penetrating injuries damage the brain. Several studies have implicated either membrane poration or ion channel dysfunction pursuant to neuronal cell death as the primary mechanism of injury. We hypothesized that traumatic stimulation of integrins may be an important etiological contributor to mild Traumatic Brain Injury. In order to study the effects of forces at the cellular level, we utilized two hierarchical, in vitro systems to mimic traumatic injury to rat cortical neurons: a high velocity stretcher and a magnetic tweezer system. In one system, we controlled focal adhesion formation in neurons cultured on a stretchable substrate loaded with an abrupt, one dimensional strain. With the second system, we used magnetic tweezers to directly simulate the abrupt injury forces endured by a focal adhesion on the neurite. Both systems revealed variations in the rate and nature of neuronal injury as a function of focal adhesion density and direct integrin stimulation without membrane poration. Pharmacological inhibition of calpains did not mitigate the injury yet the inhibition of Rho-kinase immediately after injury reduced axonal injury. These data suggest that integrin-mediated activation of Rho may be a contributor to the diffuse axonal injury reported in mild Traumatic Brain Injury.

Vascular smooth muscle contractility depends on cell shape

The physiologic role of smooth muscle structure in defining arterial function is poorly understood. We aimed to elucidate the relationship between vascular smooth muscle architecture and functional contractile output. Using microcontact printing and muscular thin film technology, we engineered in vitro vascular tissues with strictly defined geometries and tested their contractile function. In all tissues, vascular smooth muscle cells (VSMCs) were highly aligned with in vivo-like spindle architecture, and contracted physiologically in response to stimulation with endothelin-1. However, tissues wherein the VSMCs were forced into exaggerated spindle elongation exerted significantly greater contraction force per unit cross-sectional area than those with smaller aspect ratios. Moreover, this increased contraction did not occur in conjunction with an increase in traditionally measured contractile phenotype markers. These results suggest that cellular architecture within vascular tissues plays a significant role in conferring tissue function and that, in some systems, traditional phenotype characterization is not sufficient to define a functionally contractile population of VSMCs.

Computational study of growth and remodelling in the aortic arch

Opening angles (OAs) are associated with growth and remodelling in arteries. One curiosity has been the relatively large OAs found in the aortic arch of some animals. Here, we use computational models to explore the reasons behind this phenomenon. The artery is assumed to contain a smooth muscle/collagen phase and an elastin phase. In the models, growth and remodelling of smooth muscle/collagen depends on wall stress and fluid shear stress. Remodelling of elastin, which normally turns over very slowly, is neglected. The results indicate that OAs generally increase with longitudinal curvature (torus model), earlier elastin production during development, and decreased wall stiffness. Correlating these results with available experimental data suggests that all of these effects may contribute to the large OAs in the aortic arch. The models also suggest that the slow turnover rate of elastin limits longitudinal growth. These results should promote increased understanding of the causes of residual stress in arteries.

Regional epicardial strain in the embryonic chick heart during the early looping stages

Epicardial strains were measured in Hamburger-Hamilton stage 11 and 12 embryonic chick hearts (1.6-2.0 days of incubation). These stages include part of the early phase of cardiac looping, as the initially straight heart tube bends and twists to form a curved c-shaped tube. By analyzing the motion of microbeads placed on the myocardial surface, we measured strains near the outer curvature, in the central region, and near the inner curvature of the primitive ventricle. No significant differences in strain were found between stages. Relative to end diastole, all three regions shortened by about 10% during systole in the circumferential direction, and the outer curvature shortened longitudinally by about 5%. In contrast, and unlike strains in older hearts, the inner curvature and central regions elongated by approximately 5-10% in the longitudinal direction during systole. These results are consistent with microstructural data and suggest that the material properties of the outer curvature are relatively isotropic, whereas the properties of the central and inner curvature regions are orthotropic, with contractile stress exerted primarily in the circumferential direction.

The contractile strength of vascular smooth muscle myocytes is shape dependent

Vascular smooth muscle cells in muscular arteries are more elongated than those in elastic arteries. Previously, we reported changes in the contractility of engineered vascular smooth muscle tissue that appeared to be correlated with the shape of the constituent cells, supporting the commonly held belief that elongated muscle geometry may allow for the better contractile tone modulation required in response to changes in blood flow and pressure. To test this hypothesis more rigorously, we developed an in vitro model by engineering human vascular smooth muscle cells to take on the same shapes as those seen in elastic and muscular arteries and measured their contraction during stimulation with endothelin-1. We found that in the engineered cells, actin alignment and nuclear eccentricity increased as the shape of the cell elongated. Smooth muscle cells with elongated shapes exhibited lower contractile strength but greater percentage increase in contraction after endothelin-1 stimulation. We analysed the relationship between smooth muscle contractility and subcellular architecture and found that changes in contractility were correlated with actin alignment and nuclear shape. These results suggest that elongated smooth muscle cells facilitate muscular artery tone modulation by increasing its dynamic contractile range.

Smooth Muscle Phenotype Switching in Blast Traumatic Brain Injury-Induced Cerebral Vasospasm

Due to increased survival rates among soldiers exposed to explosive blasts, blast-induced traumatic brain injury (bTBI) has become much more prevalent in recent years. Cerebral vasospasm (CVS) is a common manifestation of brain injury whose incidence is significantly increased in bTBI. CVS is characterized by initial vascular smooth muscle cell (VSMC) hypercontractility, followed by prolonged vessel remodeling and lumen occlusion, and is traditionally associated with subarachnoid hemorrhage (SAH), but recent results suggest that mechanical injury during bTBI can cause mechanotransduced VSMC hypercontractility and phenotype switching necessary for CVS development, even in the absence of SAH. Here, we review the mechanisms by which mechanical stimulation and SAH can synergistically drive CVS progression, complicating treatment options in bTBI patients.

Long-term vascular contractility assay using genipin-modified muscular thin films.

Vascular disease is a leading cause of death globally and typically manifests chronically due to long-term maladaptive arterial growth and remodeling. To date, there is no in vitro technique for studying vascular function over relevant disease time courses that both mimics in vivo-like tissue structure and provides a simple readout of tissue stress. We aimed to extend tissue viability in our muscular thin film contractility assay by modifying the polydimethylsiloxane (PDMS) substrate with micropatterned genipin, allowing extracellular matrix turnover without cell loss. To achieve this, we developed a microfluidic delivery system to pattern genipin and extracellular matrix proteins on PDMS prior to cell seeding. Tissues constructed using this method showed improved viability and maintenance of in vivo-like lamellar structure. Functional contractility of tissues fabricated on genipin-modified substrates remained consistent throughout two weeks in culture. These results suggest that muscular thin films with genipin-modified PDMS substrates are a viable method for conducting functional studies of arterial growth and remodeling in vascular diseases.