Christopher J. Pino

BVES regulates EMT in human corneal and colon cancer cells and is silenced via promoter methylation in human colorectal carcinoma

The acquisition of a mesenchymal phenotype is a critical step in the metastatic progression of epithelial carcinomas. Adherens junctions (AJs) are required for suppressing this epithelial-mesenchymal transition (EMT) but less is known about the role of tight junctions (TJs) in this process. Here, we investigated the functions of blood vessel epicardial substance (BVES, also known as POPDC1 and POP1), an integral membrane protein that regulates TJ formation. BVES was found to be underexpressed in all stages of human colorectal carcinoma (CRC) and in adenomatous polyps, indicating its suppression occurs early in transformation. Similarly, the majority of CRC cell lines tested exhibited decreased BVES expression and promoter DNA hypermethylation, a modification associated with transcriptional silencing. Treatment with a DNA-demethylating agent restored BVES expression in CRC cell lines, indicating that methylation represses BVES expression. Reexpression of BVES in CRC cell lines promoted an epithelial phenotype, featuring decreased proliferation, migration, invasion, and anchorage-independent growth; impaired growth of an orthotopic xenograft; and blocked metastasis. Conversely, interfering with BVES function by expressing a dominant-negative mutant in human corneal epithelial cells induced mesenchymal features. These biological outcomes were associated with changes in AJ and TJ composition and related signaling. Therefore, BVES prevents EMT, and its epigenetic silencing may be an important step in promoting EMT programs during colon carcinogenesis.

Bioartificial Renal Epithelial Cell System (BRECS): A Compact, Cryopreservable Extracorporeal Renal Replacement Device.

Renal cell therapy has shown clinical efficacy in the treatment of acute renal failure (ARF) and promise for treatment of end-stage renal disease (ESRD) by supplementing conventional small solute clearance (hemodialysis or hemofiltration) with endocrine and metabolic function provided by cells maintained in an extracorporeal circuit. A major obstacle in the widespread adoption of this therapeutic approach is the lack of a cryopreservable system to enable distribution, storage, and therapeutic use at point of care facilities. This report details the design, fabrication, and assessment of a Bioartificial Renal Epithelial Cell System (BRECS), the first all-in-one culture vessel, cryostorage device, and cell therapy delivery system. The BRECS was loaded with up to 20 cell-seeded porous disks, which were maintained by perfusion culture. Once cells reached over 5 × 106 cells/disk for a total therapeutic dose of approximately 108 cells, the BRECS was cryopreserved for storage at -80°C or -140°C. The BRECS was rapidly thawed, and perfusion culture was resumed. Near precryopreservation values of cell viability, metabolic activity, and differentiated phenotype of functional renal cells were confirmed post-reconstitution. This technology could be extended to administer other cell-based therapies where metabolic, regulatory, or secretion functions can be leveraged in an immunoisolated extracorporeal circuit.

A Biomimetic Membrane Device That Modulates the Excessive Inflammatory Response to Sepsis

Objective Septic shock has a clinical mortality rate approaching fifty percent. The major clinical manifestations of sepsis are due to the dysregulation of the hosts response to infection rather than the direct consequences of the invading pathogen. Central to this initial immunologic response is the activation of leukocytes and microvascular endothelium resulting in cardiovascular instability, lung injury and renal dysfunction. Due to the primary role of leukocyte activation in the sepsis syndrome, a synthetic biomimetic membrane, called a selective cytopheretic device (SCD), was developed to bind activated leukocytes. The incorporation of the SCD along an extracorporeal blood circuit coupled with regional anticoagulation with citrate to lower blood ionized calcium was devised to modulate leukocyte activation in sepsis. Design Laboratory investigation. Setting University of Michigan Medical School. Subjects Pigs weighing 30-35 kg. Interventions To assess the effect of the SCD in septic shock, pigs were administered 30×1010 bacteria/kg body weight of Escherichia coli into the peritoneal cavity and within 1 hr were immediately placed in an extracorporeal circuit containing SCD. Measurements and Main Results In this animal model, the SCD with citrate compared to control groups without the SCD or with heparin anticoagulation ameliorated the cardiovascular instability and lung sequestration of activated leukocytes, reduced renal dysfunction and improved survival time compared to various control groups. This effect was associated with minimal elevations of systemic circulating neutrophil activation. Conclusions These preclinical studies along with two favorable exploratory clinical trials form the basis of an FDA-approved investigational device exemption for a pivotal multicenter, randomized control trial currently underway.

Bves modulates tight junction associated signaling.

Blood vessel epicardial substance (Bves) is a transmembrane adhesion protein that regulates tight junction (TJ) formation in a variety of epithelia. The role of TJs within epithelium extends beyond the mechanical properties. They have been shown to play a direct role in regulation of RhoA and ZONAB/DbpA, a y-box transcription factor. We hypothesize that Bves can modulate RhoA activation and ZONAB/DbpA activity through its regulatory effect on TJ formation. Immortalized human corneal epithelial (HCE) cells were stably transfected with Flag-tagged full length chicken Bves (w-Bves) or C-terminus truncated Bves (t-Bves). We found that stably transfected w-Bves and t-Bves were interacting with endogenous human Bves. However, interaction with t-Bves appeared to disrupt cell membrane localization of endogenous Bves and interaction with ZO-1. w-Bves cells exhibited increased TJ function reflected by increased trans-epithelial electrical resistance, while t-Bves cells lost TJ protein immunolocalization at cell-cell contacts and exhibited decreased trans-epithelial electrical resistance. In parental HCE and w-Bves cells ZONAB/DbpA and GEF-H1 were seen at cell borders in the same pattern as ZO-1. However, expression of t-Bves led to decreased membrane localization of both ZONAB/DbpA and GEF-H1. t-Bves cells had increased RhoA activity, as indicated by a significant 30% increase in FRET activity compared to parental HCE cells. ZONAB/DbpA transcriptional activity, assessed using a luciferase reporter probe, was increased in t-Bves cells. These studies demonstrate that Bves expression and localization can regulate RhoA and ZONAB/DbpA activity.

Seeding of Corneal Wounds by Epithelial Cell Transfer from Micropatterned PDMS Contact Lenses

Persistent corneal wounds result from numerous eye disorders, and to date, available treatments often fail to accelerate reepithelialization, the key initial step in wound healing. To speed reepithelialization, we explored a cell-transfer transplant method utilizing polydimethylsiloxane (PDMS) contact lenses to deliver epithelial cells derived from limbal explants directly within a corneal wound. Human primary epithelial cells and an immortalized corneal epithelial cell line (HCE-SV40) grew well on PDMS contact lenses and their morphology and growth rates where similar to cells grown on tissue culture polystyrene. To initially study cell transfer from PDMS, HCE-SV40 cells were seeded onto PDMS with or without micropatterned posts. After a day in culture, HCE-SV40 cells attached to the unpatterned PDMS uniformly, whereas on micropatterned PDMS they appeared to attach primarily between posts. The cell-covered PDMS contacts were then placed cell-side down onto tissue culture plastic and, after 1, 2, or 3 days, the PDMS contact was removed and the transferred cells were trypsinized and counted. Micropatterned PDMS contact lenses with 100-μm-diameter posts and a post height of 40 μm transferred three times as many cells as unpatterned PDMS. Cell transfer to a wounded cornea was tested in a pig cornea organ culture model deepithelialized by alkali treatment. Post micropatterned PDMS contact lenses were seeded with labeled HCE-SV40 cells at a density 50,000 cells/cm2 and applied to the wounded pig corneas. After 24, 48, or 96 h of application, PDMS contact lenses were removed, corneas fixed with formaldehyde, and sectioned. After 48 h, epithelial cells transferred from post micropatterned contact lenses to provide 35% epithelial coverage of denuded pig corneas; after 96 h coverage was 65%. We conclude that cell transfer from epithelial-coated PDMS contact lenses micropatterned with posts provides a promising approach to reepithelialize corneal surfaces.

Cell-based approaches for the treatment of systemic inflammation

Acute and chronic solid organ failures are costly disease processes with high mortality rates. Inflammation plays a central role in both acute and chronic organ failure, including heart, lung and kidney. In this regard, new therapies for these disorders have focused on inhibiting the mediators of inflammation, including cytokines and free radicals, with little or no success in clinical studies. Recent novel treatment strategies have been directed to cell-based rather than mediator-based approaches, designed to immunomodulate the deleterious effects of inflammation on organ function. One approach, cell therapy, replaces cells that were damaged in the acute or chronic disease process with stem/progenitor technology, to rebalance excessive inflammatory states. As an example of this approach, the use of an immunomodulatory role of renal epithelial progenitor cells to treat acute renal failure (ARF) and multiorgan failure arising from acute kidney injury is reviewed. A second therapeutic pathway, cell processing, does not incorporate stem/progenitor cells in the device, but rather biomimetic materials that remove and modulate the primary cellular components, which promote the worsening organ tissue injury associated with inflammation. The use of an immunomodulating leukocyte selective cytopheretic inhibitory device is also reviewed as an example of this cell processing approach. Both of these unconventional strategies have shown early clinical efficacy in pilot clinical trials and may transform the therapeutic approach to organ failure disorders.

Cell-Based Strategies for the Treatment of Kidney Dysfunction: A Review

Conventional treatment of acute and chronic renal diseases has focused on solute removal. Novel strategies aim to treat the multifactorial disease states of acute kidney injury and chronic kidney disease by mitigating inflammation. Cell-based technologies for the treatment of kidney dysfunction fall under two broad categories: cell therapy and cell processing. Cell therapy utilizes cells that are isolated, cultured outside of the body, and reintroduced as therapy, leveraging beneficial metabolic and synthetic functions. For example, renal tubule cells have been used to provide gluconeogenesis, ammoniagenesis, metabolism of glutathione, catabolism of important peptide hormones, growth factors, and cytokines critical to multiorgan homeostasis and immunomodulation to treat renal dysfunction. Cell processing focuses on altering the characteristics of cell populations inside the body to provide therapy. The selective cytopheretic device is an example of this novel therapeutic strategy that aims to modulate the innate immune response during organ dysfunction, additional organ injury, by binding and deactivating leukocytes. In this review, both cell therapy and cell processing approaches will be discussed in the context of acute kidney injury and chronic renal disease.

A bio-artificial renal epithelial cell system conveys survival advantage in a porcine model of septic shock.

Renal cell therapy using the hollow fiber based renal assist device (RAD) improved survival time in an animal model of septic shock (SS) through the amelioration of cardiac and vascular dysfunction. Safety and ability of the RAD to improve clinical outcomes was demonstrated in a Phase II clinical trial, in which patients had high prevalence of sepsis. Even with these promising results, clinical delivery of cell therapy is hampered by manufacturing hurdles, including cell sourcing, large‐scale device manufacture, storage and delivery. To address these limitations, the bioartificial renal epithelial cell system (BRECS) was developed. The BRECS contains human renal tubule epithelial cells derived from adult progenitor cells using enhanced propagation techniques. Cells were seeded onto trabeculated disks of niobium‐coated carbon, held within cryopreservable, perfusable, injection‐moulded polycarbonate housing. The study objective was to evaluate the BRECS in a porcine model of SS to establish conservation of efficacy after necessary cell sourcing and design modifications; a pre‐clinical requirement to move back into clinical trials. SS was incited by peritoneal injection of E. coli simultaneous to insertion of BRECS (n=10) or control (n=15), into the ultrafiltrate biofeedback component of an extracorporeal circuit. Comparable to RAD, prolonged survival of the BRECS cohort was conveyed through stabilization of cardiac output and vascular leak. In conclusion, the demonstration of conserved efficacy with BRECS therapy in a porcine SS model represents a crucial step toward returning renal cell therapy to the clinical setting, initially targeting ICU patients with acute kidney injury requiring continuous renal replacement therapy. Copyright

Development of a wearable bioartificial kidney using the Bioartificial Renal Epithelial Cell System (BRECS).

Cell therapy for the treatment of renal failure in the acute setting has proved successful, with therapeutic impact, yet development of a sustainable, portable bioartificial kidney for treatment of chronic renal failure has yet to be realized. Challenges in maintaining an anticoagulated blood circuit, the typical platform for solute clearance and support of the biological components, have posed a major hurdle in advancement of this technology. This group has developed a Bioartificial Renal Epithelial Cell System (BRECS) capable of differentiated renal cell function while sustained by body fluids other than blood. To evaluate this device for potential use in end‐stage renal disease, a large animal model was established that exploits peritoneal dialysis fluid for support of the biological device and delivery of cell therapy while providing uraemic control. Anephric sheep received a continuous flow peritoneal dialysis (CFPD) circuit that included a BRECS. Sheep were treated with BRECS containing 1 × 108 renal epithelial cells or acellular sham devices for up to 7 days. The BRECS cell viability and activity were maintained with extracorporeal peritoneal fluid circulation. A systemic immunological effect of BRECS therapy was observed as cell‐treated sheep retained neutrophil oxidative activity better than sham‐treated animals. This model demonstrates that use of the BRECS within a CFPD circuit embodies a feasible approach to a sustainable and effective wearable bioartificial kidney. Copyright

Surface Functionality as a Means to Impact Polymer Nanoparticle Size and Structure

When polymeric nanoparticles (NPs) are formed by nanoprecipitation, which is a nucleation-growth process, the control over size requires changing the polymer concentration or solvent composition. Here, we demonstrate that the NP size can be controlled independent of polymer variables by introducing a polyelectrolyte (PE) in the aqueous phase. PEs that exhibit hydrogen bonding (H-bonding) yield a reduction in NP size, whereas PEs that do not possess this characteristic promote the formation of larger NPs. The observed effect can be attributed to the formation of a diffusional barrier around the NP in the form of a dense shell. This principle of controlling NP size is not limited to polymers and can also be employed in the production of lipid NPs.