Publications

and urethro-vaginal (UVF) fistula are infrequently encountered in current UK practice. We reviewed the outcome of 26 consecutive patients treated in a 6-year period.

Loss of muscle stem cell (MuSC) self-renewal with aging reflects a combination of influences from the intracellular (e.g., post-transcriptional modifications) and extracellular (e.g., matrix stiffness) environment. Whereas conventional single cell analyses have revealed valuable insights into factors contributing to impaired self-renewal with age, most are limited by static measurements that fail to capture nonlinear dynamics. Using bioengineered matrices mimicking the stiffness of young and old muscle, we showed that while young MuSCs were unaffected by aged matrices, old MuSCs were phenotypically rejuvenated by young matrices. Dynamical modeling of RNA velocity vector fields <i>in silico</i> revealed that soft matrices promoted a self-renewing state in old MuSCs by attenuating RNA decay. Vector field perturbations demonstrated that the effects of matrix stiffness on MuSC self-renewal could be circumvented by fine-tuning the expression of the RNA decay machinery. These results demonstrate that post-transcriptional dynamics dictate the negative effect of aged matrices on MuSC self-renewal.

Stress urinary incontinence (SUI) is the involuntary release of urine during sudden increases in abdominal pressures. SUI is common in women after vaginal delivery or pelvic trauma and may alter the biomechanical properties of the urethra. Thus we hypothesize that injury due to vaginal distension (VD) decreases urethral basal tone and passive stiffness. This study aimed to assess the biomechanical properties of the urethra after VD in the baseline state, where basal muscle tone and extracellular matrix (ECM) are present, and in the passive state, where inactive muscle and ECM are present. Female rat urethras were isolated in a rat model of acute SUI induced by simulated birth trauma. Our established ex vivo system was utilized, wherein we applied intraluminal static pressures ranging from 0 to 20 mmHg. Outer diameter was measured via a laser micrometer. Measurements were recorded via computer. Urethral thickness was assessed histologically. Stress-strain responses of the urethra were altered by VD. Quantification of biomechanical parameters indicated that VD decreased baseline stiffness. The passive peak incremental elastic modulus of the distal segment in VD urethras was less than for controls (1.84 +/- 0.67 vs. 1.19 +/- 0.70 x 10(6) dyne/cm(2), respectively; P = 0.016). An increase was noted in passive low-pressure compliance values in proximal VD urethras compared with controls (9.44 +/- 2.43 vs. 4.62 +/- 0.60 mmHg(-1), respectively; P = 0.04). Biomechanical analyses suggest that VD alters urethral basal tone, proximal urethral compliance, and distal stiffness. Lack of basal smooth muscle tone, in combination with these changes in the proximal and distal urethra, may contribute to SUI induced by VD.

The autogenous saphenous vein remains the graft of choice for both coronary (500,000 annually in the US)1 or peripheral (80,000 annually)2 arterial bypass procedures. Failure of arterial vein grafts (AVGs) remains a major problem, and patients with failed grafts will die or require re-operation. Intimal hyperplasia (IH) accounts for 20% to 40% of all AVG failures3. It is believed that this adverse pathological response by AVGs is largely due to their abrupt exposure to the significantly elevated circumferential wall stress (CWS) associated with the arterial system4. We believe that if an AVG is given an ample opportunity to adapt and remodel to the stresses of its new environment, cellular injury may be reduced, thus limiting the initiating mechanisms of IH. The goal of this work was to develop a new mechanical conditioning paradigm, in the form of a peri-adventitially placed, biodegradable polymer wrap, to safely and functionally “arterialize” AVGs in situ. The electrospun polymer wrap was tuned so that as it degraded over a desired period of time, the mechanical support offered by it was reduced gradually, and hence the vein was exposed to slowly increasing levels of CWS in situ.

Mechanical forces have been shown to be important stimuli for the determination and maintenance of cellular phenotype and function. Many cells are constantly exposed in vivo to cyclic pressure, shear stress, and/or strain. Therefore, the ability to study the effects of these stimuli in vitro is important for understanding how they contribute to both normal and pathologic states. While there exist commercial as well as custom-built devices for the extended application of cyclic strain and shear stress, very few cyclic pressure systems have been reported to apply stimulation longer than 48 h. However, pertinent responses of cells to mechanical stimulation may occur later than this. To address this limitation, we have designed a new cyclic hydrostatic pressure system based upon the following design variables: minimal size, stability of pressure and humidity, maximal accessibility, and versatility. Computational fluid dynamics (CFD) was utilized to predict the pressure and potential shear stress within the chamber during the first half of a 1.0 Hz duty cycle. To biologically validate our system, we tested the response of bone marrow progenitor cells (BMPCs) from Sprague Dawley rats to a cyclic pressure stimulation of 120/80 mm Hg, 1.0 Hz for 7 days. Cellular morphology was measured using Scion Image, and cellular proliferation was measured by counting nuclei in ten fields of view. CFD results showed a constant pressure across the length of the chamber and no shear stress developed at the base of the chamber where the cells are cultured. BMPCs from Sprague Dawley rats demonstrated a significant change in morphology versus controls by reducing their size and adopting a more rounded morphology. Furthermore, these cells increased their proliferation under cyclic hydrostatic pressure. We have demonstrated that our system imparts a single mechanical stimulus of cyclic hydrostatic pressure and is capable of at least 7 days of continuous operation without affecting cellular viability. Furthermore, we have shown for the first time that BMPCs respond to cyclic hydrostatic pressure by alterations in morphology and increased proliferation.

Objective Elastogenesis within the medial layer of the aortic wall involves a cascade of events orchestrated primarily by smooth muscle cells, including transcription of elastin and a cadre of elastin chaperone matricellular proteins, deposition and cross-linking of tropoelastin coacervates, and maturation of extracellular matrix fiber structures to form mechanically competent vascular tissue. Elastic fiber disruption is associated with aortic aneurysm; in aneurysmal disease a thin and weakened wall leads to a high risk of rupture if left untreated, and non-surgical treatments for small aortic aneurysms are currently limited. This study analyzed the effect of adipose-derived stromal cell secreted factors on each step of the smooth muscle cell elastogenesis cascade within a three-dimensional fibrin gel culture platform. Approach and results We demonstrate that adipose-derived stromal cell secreted factors induce an increase in smooth muscle cell transcription of tropoelastin, fibrillin-1, and chaperone proteins fibulin-5, lysyl oxidase, and lysyl oxidase-like 1, formation of extracellular elastic fibers, insoluble elastin and collagen protein fractions in dynamically-active 30-day constructs, and a mechanically competent matrix after 30 days in culture. Conclusion Our results reveal a potential avenue for an elastin-targeted small aortic aneurysm therapeutic, acting as a supplement to the currently employed passive monitoring strategy. Additionally, the elastogenesis analysis workflow explored here could guide future mechanistic studies of elastin formation, which in turn could lead to new non-surgical treatment strategies.

The primary goal of tissue engineering is to develop a biological, mechanically-robust, and anti-thrombogenic vascular graft to replace diseased or damaged tissue and organs [1]. For example, researchers have incorporated smooth muscle cells (SMCs) into extracellular matrix to provide a living, functional conduits with the intended purpose of replacing SMC-containing tubes, such as the blood vessel, urethra, esophagus, intestine, etc. Although the preferred source is autologous cells to avoid immunological rejection, adult SMCs are difficult to obtain and expand. An alternative source of autologous cells could be bone marrow derived stem cells (BMSCs), which differentiate toward mesenchymal and hematopoietic lineages [2].

AAA rupture occurs when wall stresses exceed the wall strength. We tested two hypotheses: 1) the AAA wall near thick intraluminal thrombus (ILT) exhibits impaired remodeling compared to wall near thin ILT and 2) macrophages (mΦ) cultured under hypoxic conditions exhibit altered enzyme expression compared to mΦ in normoxic conditions. For 1, pairs of AAA wall, selected based on disparate thicknesses of near ILT, were snap frozen. QPCR was used to compare the gene expression. Total wall thickness was measured on H&amp;E sections. For 2, peripheral mononuclear cells were isolated from human buffy coats and cultured under hypoxic (2% O 2 ) or normoxic (20% O 2 ) conditions in serum‐free medium. Media levels of MMP‐9 were determined using ELISA. We found a down‐regulation of MMP‐2, MMP‐9, uPA, tPA, CD68, and collagen IV, and an up‐regulation of HO‐1, ORP‐150, ICAM‐1, IL‐12, IFN‐γ, and iNOS in AAA wall near thick ILT compared to AAA wall near thin ILT. AAA wall thickness was positively correlated (ρ=0.476) to ILT thickness. MΦ cultured in hypoxic conditions showed a marked decrease in secreted MMP‐9 activity and protein levels relative to mΦ cultured in normoxic conditions on collagen I or fibrin. The results support hypothesis 1, suggesting that compared to AAA wall near thin ILT, the AAA wall adjacent to a thick ILT has a more activated immune component and impaired remodeling mechanisms. Our results also suggest that the hypoxic environment of the AAA wall may lead to an alteration in protein expression by mΦ. This work was supported by NIH R01‐HL79313.

Abdominal aortic aneurysm (AAA) intervention and surveillance is currently based on maximum transverse diameter, even though it is recognized that this might not be the best strategy. About 10% of patients with small AAA transverse diameters, for whom intervention is not considered, still rupture; while patients with large AAA transverse diameters, for whom intervention would have been recommended, have stable aneurysms that do not rupture. While maximum transverse diameter is easy to measure and track in clinical practice, one of its main drawbacks is that it does not represent the whole AAA and rupture seldom occurs in the region of maximum transverse diameter. By following maximum transverse diameter alone clinicians are missing information on the shape change dynamics of the AAA, and clues that could lead to better patient care. We propose here a method to register AAA surfaces that were obtained from the same patient at different time points. Our registration method could be used to track the local changes of the patient-specific AAA. To achieve registration, our procedure uses a consistent parameterization of the AAA surfaces followed by strain relaxation. The main assumption of our procedure is that growth of the AAA occurs in such a way that surface strains are smoothly distributed, while regions of small and large surface growth can be differentiated. The proposed methodology has the potential to unravel different patterns of AAA growth that could be used to stratify patient risks.

One of the major limitations in tissue engineering is cell sourcing. Multipotent progenitor cells appear to have many promising features for that purpose. Mechanical stimulation is known to play an important role in determining cell phenotype. The aim of this work was to investigate the effects of cyclic stretch on rat bone marrow derived progenitor cell (BMPC) morphology and smooth muscle-directed differentiation within a three-dimensional fibrin matrix. BMPCs were suspended in a fibrin gel, pipetted into the trough of Flexcell Tissue-Train plates, and stimulated with 10% longitudinal cyclic stretch at 1 Hz for 6 days. Unconstrained (stress- and strain-free) and static anchored (constrained but not stretched) samples were used as controls. Stress filament area per cell was increased in the stretched samples compared to static anchored and free-float controls. Cells in the free float controls were randomly aligned, while they aligned parallel to the direction of the stress or strain in the other groups. Immunofluorescence suggested an increased expression of smooth muscle markers (smooth muscle alpha actin and h1-calponin) in both stretched and constrained control samples, but not in unconstrained controls. Qualitative assessment suggested that collagen production was increased in both mechanically stimulated samples. Proliferation was inhibited in stretched samples compared to the constrained controls. This work suggests an ability of rat BMPCs to differentiate toward a smooth-muscle-cell-like lineage when exposed to biomechanical stimulation in a three-dimensional model. The observation that the constrained samples induced changes in BMPCs suggests that stress alone may be stimulatory, but addition of cyclic stretch appears to augment the responses.

Abdominal aortic aneurysm (AAA), a localized dilation of the infrarenal aorta, represents a significant disease in the western population. There are approximately 200,000 patients in the US and 500,000 patients worldwide diagnosed with AAAs every year (Bosch, et al. 2001), and rupture of AAAs currently ranks as the 13th leading cause of death in the US. (Silverberg and Lubera 1987) In the past 30 years, the diagnosis of AAA has tripled in the Western world, and this will likely increase in the coming years as the average age of the population is increasing. (Bosch, et al. 2001)

Failure of veins employed as arterial bypass grafts via intimal hyperplasia (IH) often occurs within 5 years after implantation, requiring re-operation in 60% of all cases1. IH is characterized by de-adhesion, followed by migration of medial and adventitial smooth muscle cells (SMCs) and myofibroblasts into the intima, where they demonstrate uncontrolled proliferation. It is thought that this process may be induced by the abrupt exposure of the veins to the dynamic mechanical environment of the arterial circulation2. Veins are much thinner walled and more distensible than arteries. Therefore, the SMCs within the vein wall are exposed to significantly higher levels of stress and strain than they are accustomed2. The tissue responds to this perceived injury by thickening, which is thought to be an attempt to return the stress and strain to venous levels. However, when this response is uncontrolled it can over-compensate, leading to stenosis instead of the desired thickening or “arterialization” of the vein segment. Cellular de-adhesion, which refers to a change from a state of stronger adherence to a state of weaker adherence, is involved in the earliest response and therefore was the focus of this study. While there are many important proteins involved in the regulation of cellular adhesion, we focus our attention here to matricellular proteins, which function as adaptors and modulators of cell-matrix interactions3,4, and intracellular adhesion proteins, which have been shown to localize to cellular focal adhesion sites5,6. Tenascin-C (TN-C), thrombospondin-1,2 (TSP), and secreted protein acidic and rich in cysteine (SPARC) are matricellular proteins that exhibit highly regulated expression during development and cellular injury7. Mitogen inducible gene 2 (Mig-2) and integrin linked kinase (ILK) are intracellular proteins involved in cellular shape modulation5 and integrin-mediated signal transduction8, respectively. It is well known that many intracellular and extracellular matrix proteins are regulated by mechanical stress9,10. The purpose of this work was to explore the hypothesis that intact vein segments exposed to arterial hemodynamics will alter their expression of TN-C, TSP, SPARC, Mig-2 and ILK within 24 hours. This may induce a modulation of the level of cell adhesion, which could contribute to IH.