Venkat M. Ramakrishnan

Generation of a functional liver tissue mimic using adipose stromal vascular fraction cell-derived vasculatures

One of the major challenges in cell implantation therapies is to promote integration of the microcirculation between the implanted cells and the host. We used adipose-derived stromal vascular fraction (SVF) cells to vascularize a human liver cell (HepG2) implant. We hypothesized that the SVF cells would form a functional microcirculation via vascular assembly and inosculation with the host vasculature. Initially, we assessed the extent and character of neovasculatures formed by freshly isolated and cultured SVF cells and found that freshly isolated cells have a higher vascularization potential. Generation of a 3D implant containing fresh SVF and HepG2 cells formed a tissue in which HepG2 cells were entwined with a network of microvessels. Implanted HepG2 cells sequestered labeled LDL delivered by systemic intravascular injection only in SVF-vascularized implants demonstrating that SVF cell-derived vasculatures can effectively integrate with host vessels and interface with parenchymal cells to form a functional tissue mimic.

Dissecting the Role of Human Embryonic Stem Cell–Derived Mesenchymal Cells in Human Umbilical Vein Endothelial Cell Network Stabilization in Three-Dimensional Environments

The microvasculature is principally composed of two cell types: endothelium and mural support cells. Multiple sources are available for human endothelial cells (ECs) but sources for human microvascular mural cells (MCs) are limited. We derived multipotent mesenchymal progenitor cells from human embryonic stem cells (hES-MC) that can function as an MC and stabilize human EC networks in three-dimensional (3D) collagen-fibronectin culture by paracrine mechanisms. Here, we have investigated the basis for hES-MC-mediated stabilization and identified the pleiotropic growth factor hepatocyte growth factor/scatter factor (HGF/SF) as a putative hES-MC-derived regulator of EC network stabilization in 3D in vitro culture. Pharmacological inhibition of the HGF receptor (Met) (1 μm SU11274) inhibits EC network formation in the presence of hES-MC. hES-MC produce and release HGF while human umbilical vein endothelial cells (HUVEC) do not. When HUVEC are cultured alone the networks collapse, but in the presence of recombinant human HGF or conditioned media from human HGF-transduced cells significantly more networks persist. In addition, HUVEC transduced to constitutively express human HGF also form stable networks by autocrine mechanisms. By enzyme-linked immunosorbent assay, the coculture media were enriched in both angiopoietin-1 (Ang1) and angiopoietin-2 (Ang2), but at significantly different levels (Ang1=159±15 pg/mL vs. Ang2=30,867±2685 pg/mL) contributed by hES-MC and HUVEC, respectively. Although the coculture cells formed stabile network architectures, their morphology suggests the assembly of an immature plexus. When HUVEC and hES-MC were implanted subcutaneously in immune compromised Rag1 mice, hES-MC increased their contact with HUVEC along the axis of the vessel. This data suggests that HUVEC and hES-MC form an immature plexus mediated in part by HGF and angiopoietins that is capable of maturation under the correct environmental conditions (e.g., in vivo). Therefore, hES-MC can function as microvascular MCs and may be a useful cell source for testing EC-MC interactions.

Restoration of Physiologically Responsive Low-Density Lipoprotein Receptor-Mediated Endocytosis in Genetically Deficient Induced Pluripotent Stem Cells.

Acquiring sufficient amounts of high-quality cells remains an impediment to cell-based therapies. Induced pluripotent stem cells (iPSC) may be an unparalleled source, but autologous iPSC likely retain deficiencies requiring correction. We present a strategy for restoring physiological function in genetically deficient iPSC utilizing the low-density lipoprotein receptor (LDLR) deficiency Familial Hypercholesterolemia (FH) as our model. FH fibroblasts were reprogrammed into iPSC using synthetic modified mRNA. FH-iPSC exhibited pluripotency and differentiated toward a hepatic lineage. To restore LDLR endocytosis, FH-iPSC were transfected with a 31 kb plasmid (pEHZ-LDLR-LDLR) containing a wild-type LDLR (FH-iPSC-LDLR) controlled by 10 kb of upstream genomic DNA as well as Epstein-Barr sequences (EBNA1 and oriP) for episomal retention and replication. After six months of selective culture, pEHZ-LDLR-LDLR was recovered from FH-iPSC-LDLR and transfected into Ldlr-deficient CHO-a7 cells, which then exhibited feedback-controlled LDLR-mediated endocytosis. To quantify endocytosis, FH-iPSC ± LDLR were differentiated into mesenchymal cells (MC), pretreated with excess free sterols, Lovastatin, or ethanol (control), and exposed to DiI-LDL. FH-MC-LDLR demonstrated a physiological response, with virtually no DiI-LDL internalization with excess sterols and an ~2-fold increase in DiI-LDL internalization by Lovastatin compared to FH-MC. These findings demonstrate the feasibility of functionalizing genetically deficient iPSC using episomal plasmids to deliver physiologically responsive transgenes.

Long-term biostability and bioactivity of “fibrin linked” VEGF121in vitro and in vivo

Despite major advances in understanding angiogenesis over the last few years, the ability to induce angiogenesis in ischemic wounds or larger tissue-engineering constructs remains elusive. Serious risks and limited control over dose, duration, and localization of growth factor delivery make materials-based approaches viable alternatives. In an effort to minimize passive diffusion and control the release profile of delivered growth factors, matrix properties have been engineered with regard to pore size, growth factor affinity or stable growth factor binding. Recently, fibrin or biomimetic hydrogels have been engineered towards the covalent immobilization of vascular endothelial growth factor (VEGF). Most of the studies pertaining to VEGF delivery by fibrin gel constructs have focused on characterizing release profiles, receptor activation, and the angiogenic response in vitro and in vivo. Herein we demonstrate that gels containing covalently-linked VEGF (α2PI1-8-VEGF121), compared to diffusible VEGF, elicit stronger and longer-lasting angiogenic responses in subcutaneous implants of mice. This superior angiogenic response was due to both the sustained release and significant retention of bioactivity (80%) of the delivered engineered VEGF over a 12-day period. To the best of our knowledge, this is the first report to characterize long-term matrix liberated α2PI1-8-VEGF121 bioactivity, important for future efforts in angiogenesis research.

Pediatric prostatic abscess.

Prostatic abscesses are an increasingly rare clinical entity. They are commonly caused by spread of a gram-negative urinary tract infection. Most cases reported in urologic studies have occurred in older men and are the result of bladder outlet obstruction. To date, very few cases of pediatric prostatic abscesses have been reported in published studies, and most of these occurred in neonates. We present a case of methicillin-resistant Staphylococcus aureus prostatic abscess in an adolescent and review the related data for this unusual process.

Wnt5a Regulates the Assembly of Human Adipose Derived Stromal Vascular Fraction-Derived Microvasculatures

Human adipose-derived stromal vascular fraction (hSVF) cells are an easily accessible, heterogeneous cell system that can spontaneously self-assemble into functional microvasculatures in vivo. However, the mechanisms underlying vascular self-assembly and maturation are poorly understood, therefore we utilized an in vitro model to identify potential in vivo regulatory mechanisms. We utilized passage one (P1) hSVF because of the rapid UEA1+ endothelium (EC) loss at even P2 culture. We exposed hSVF cells to a battery of angiogenesis inhibitors and found that the pan-Wnt inhibitor IWP2 produced the most significant hSVF-EC networking decrease (~25%). To determine which Wnt isoform(s) and receptor(s) may be involved, hSVF was screened by PCR for isoforms associated with angiogenesis, with only WNT5A and its receptor, FZD4, being expressed for all time points observed. Immunocytochemistry confirmed Wnt5a protein expression by hSVF. To see if Wnt5a alone could restore IWP2-induced EC network inhibition, recombinant human Wnt5a (0–150 ng/ml) was added to IWP2-treated cultures. The addition of rhWnt5a significantly increased EC network area and significantly decreased the ratio of total EC network length to EC network area compared to untreated controls. To determine if Wnt5a mediates in vivo microvascular self-assembly, 3D hSVF constructs containing an IgG isotype control, anti-Wnt5a neutralizing antibody or rhWnt5a were implanted subcutaneously for 2w in immune compromised mice. Compared to IgG controls, anti-Wnt5a treatment significantly reduced vessel length density by ~41%, while rhWnt5a significantly increased vessel length density by ~62%. However, anti-Wnt5a or rhWnt5a did not significantly affect the density of segments and nodes, both of which measure vascular complexity. Taken together, this data demonstrates that endogenous Wnt5a produced by hSVF plays a regulatory role in microvascular self-assembly in vivo. These findings also suggest that manipulating Wnt signaling could enhance control of hSVF vascularization in tissue engineering applications.

Urinary Incontinence-85: An Expanded Prostate Cancer Composite (EPIC) Score Cutoff Value for Urinary Incontinence Determined Using Long-term Functional Data by Repeated Prospective EPIC-Score Self-assessment After Radical Prostatectomy

Purpose To determine an objective cutoff value (COV) for urinary incontinence (UI) using the Expanded Prostate Cancer Composite (EPIC) score after radical prostatectomy (RP). Methods From 2004–2013, all RP patients at our institution completed the EPIC urinary domain (EPIC-UD) questionnaire preoperatively and 6 weeks; 3, 6, 9, 12, and 18 months postoperatively; and yearly thereafter. The EPIC-UD is composed of several questions, 4 of which address UI qualitatively (EPIC-UI). Furthermore, patients were asked to complete a global quality of life (QoL) questionnaire regarding continence. The EPIC COV was calculated using receiver operating characteristic (ROC) analysis. Correlations between the EPIC-UI and quantitative QoL were evaluated using the Kendall-Tau test. Results We analyzed 239 patients with a median age of 63 years (interquartile range [IQR], 59–66 years), a median follow-up of 48 months (IQR, 30–78 months) and a median preoperative EPIC-UI score of 100 (IQR, 91.75–100). The ROC analysis for the distinction between EPIC-UI and the use of ≤1 pad/day yielded an EPIC-UI COV of >85, which we termed the UI-85, with an area under the curve of 0.857 (P<0.0001). A stronger correlation was seen between QoL scores and the UI-85 (1 year postoperatively: correlation coefficient [CC], 0.592; P<0.0001) than between QoL and not using a pad (CC, 0.512; P<0.0001). Conclusions The calculated COV of the EPIC-UI for continence was 85. UI is a multidimensional condition that cannot be adequately characterized by a single piece of information, such as pad usage only. Hence, the UI-85 represents a nuanced and straightforward tool for monitoring and comparing continence between different time points and cohorts in a multidimensional and objective manner.

Angiogenesis and Vascularity for Tissue Engineering Applications

Tissue engineering is a field of medicine that has experienced significant growth in prominence over the past three decades. Though traditional interventions exist for many medical maladies, tissue engineering aims to combat such disorders through the synthesis of body tissues and organs, resulting in functional implants. Tissue engineering takes an innovative approach, often utilizing autologous stem cells for tissue construction or materials that are biocompatible while avoiding immune rejection (Nomi et al., 2002). Despite its immense successes, a major hurdle still faces tissue engineering. Large volumes of implanted tissue are unable to stimulate the formation of necessary blood vessels required for their survival. In the body, naturally occurring, equivalent vascular networks serve vital functions in gas and nutrient exchange, metabolic processes, and waste expulsion. Though individual, large vessels have been successfully engineered for implant, it is still exceptionally difficult to fashion a stable and sustainable network of vessels for large volumes of tissue (Nomi et al., 2002). Neovascularization after tissue damage requires a level of positive and negative control that has not been successfully replicated in a laboratory environment to date. As such, rapid de novo synthesis of a controlled, established vascular network remains a challenge today. Angiogenesis is the morphogenic process of forming new blood vessels from pre-existing ones (Laschke et al., 2006; Dai and Rabie, 2007; Li and Rabie, 2007). This event plays an important, normal physiological role in wound healing, tissue repair, pregnancy, and exercise (Ferrara and Davis-Smyth, 1997), and exists in contrast to vasculogenesis (the formation of the every first blood vessels in the body, and especially predominant in embryological development). Yet, the abuse of angiogenesis, leading to an uncontrolled vascular formation as a consequence of epigenetic influence, nucleotide polymorphisms, or endocrine irregularities can also result in tumor formation (Verbridge et al., 2010). However, angiogenesis is clearly an activity that is central to development and tissue maintenance. Successful modulation of angiogenesis can have profound therapeutic outcomes for organs and tissues deprived of an adequate, stable vasculature. Studies from the last two decades have shown that the manipulation of various factors directly influences angiogenic outcome.

A Prospective Analysis of the Effects of Nerve-Sparing Radical Prostatectomy on Urinary Continence Based on Expanded Prostate Cancer Index Composite and International Index of Erectile Function Scoring Systems

Purpose This study aims to objectively characterize the effect of successful nerve sparing (NS) during radical prostatectomy (RP) on postoperative urinary continence (UC) using International Index of Erectile Function (IIEF)-scores and a previously described Expanded Prostate Cancer Index Composite (EPIC) score cutoff value (COV) for UC. Several notable studies on this topic present conflicting outcomes. This is largely due to a lack of clear definitions and consensus regarding preserved erectile function (EF) and UC. Methods This study is comprised of all patients who underwent RP at the Kantonsspital Baden, Switzerland, between 2004 and 2013. Patients completed self-assessment questionnaires for UC (EPIC) and EF (IIEF) pre- and postoperatively (3, 6, 9, 12, 18, and 24 months; yearly thereafter). We used a previously described EPIC subscore COV, with “satisfactory continence” signified by a score >85. Statistical analysis was performed using Kaplan-Meier and Cox regression analyses for “surgeon-” and “IIEF-defined” NS definitions. Results Of 236 men with a median age of 63 years (interquartile range [IQR], 59–66 years) and median follow-up time of 48 months (IQR, 30–78 months), 176 underwent unilateral (n=33) or bilateral (n=143) NS RP. Fifty-four underwent non-NS (NNS) RP. Kaplan-Meier analyses identified the following risk factors for UC: age, prostate volume, cancer risk group, and NS status. In surgeon-defined NS RP cases, multivariate analysis for regaining continence demonstrated no significant difference (hazard ratio [HR], 0.78; 95% confidence interval [CI], 0.48–1.25; P=0.3). With successful IIEF-defined NS RPs, regression analysis demonstrated no significant difference (HR, 0.89; 95% CI, 0.59–1.35; P=0.58). Conclusions In our population, analysis and comparison of surgeon- and IIEF-defined NS and NNS cohorts revealed that NS RP did not improve postoperative UC. The conservation of UC alone should not motivate surgeons or patients to pursue NS RP.

209 FIBRIN TG-VEGF IS A SUPPORTER OF EARLY ANGIOGENESIS IN URINARY SPHINCTER ENGINEERING

INTRODUCTION AND OBJECTIVES: A 3-D construct seeded with autologous cells on a biodegradable scaffold could serve as tissue replacement in pelvic floor reconstruction. Our goals are to design a scaffold that incorporates suture suitable for implantation and optimizes the conditions for a seeded implant in a 3-D construct by testing various formulations of collagen and hyaluronan. METHODS: The C2C12 mouse myoblast cell line was used. Scaffold matrix components included collagen and hyaluronan (HystemHP®, Glycosan Biosystems). Constructs were seeded at 3, 4.5, and 6 10 cells/ml and polymerized in a silicone mold. 1:1,10:1 and 20:1 ratios of collagen:hyaluronan (C:H) were tested. Three scaffold designs were tested: constructs with a suture running through the middle, with Nitex mesh at the ends only, and with suture and mesh just inside the edges of the matrix. After 21 days, constructs were tested for passive stiffness. The constructs were then stained for dead cells, cell nuclei and filamentous actin, and confocal imaging was performed. RESULTS: All construct conditions resulted in elongated, differentiated muscle cells with filamentous actin that appeared fused and multinucleated. C:H at 1:1 produced a more cellular construct with a greater proportion of elongated cells and fewer dead cells at 3 weeks than the 20:1 and 10:1 (C:H) samples (Figure 1). Constructs seeded with 6 10 cells/ml and C:H of 1:1 were 2x stiffer than those seeded at lower densities. The suture-only design (tension-free) resulted in random cell aggregation. Mesh alone also resulted in cell aggregation due to lack of integration of cells into the mesh. Combining suture and mesh allowed cells to integrate into the mesh and promoted rearrangement of the cells and matrix with development of uniaxial tension between suture ends which promoted cell elongation and alignment. CONCLUSIONS: Greater cell viability was observed in constructs with an equal ratio of C:H. Combining suture with mesh located inside the matrix promoted the development of uniaxial tension that allowed the cells to align in the direction of tension. A higher seeding density resulted in a stiffer construct. Further testing is needed to determine the viability and functionality of constructs in vivo.