Specific Binding of Alzheimer's Aß Peptides to Extracellular Vesicles.

Alzheimer's disease (AD) is the fifth leading cause of death among adults aged 65 and older, yet the onset and progression of the disease is poorly understood. What is known is that the presence of amyloid, particularly polymerized Aß42, defines when people are on the AD continuum. Interestingly, as AD progresses, less Aß42 is detectable in the plasma, a phenomenon thought to result from Aß becoming more aggregated in the brain and less Aß42 and Aß40 being transported from the brain to the plasma via the CSF. We propose that extracellular vesicles (EVs) play a role in this transport. EVs are found in bodily fluids such as blood, urine, and cerebrospinal fluid and carry diverse "cargos" of bioactive molecules (e.g., proteins, nucleic acids, lipids, metabolites) that dynamically reflect changes in the cells from which they are secreted. While Aß42 and Aß40 have been reported to be present in EVs, it is not known whether this interaction is specific for these peptides and thus whether amyloid-carrying EVs play a role in AD and/or serve as brain-specific biomarkers of the AD process. To determine if there is a specific interaction between Aß and EVs, we used isothermal titration calorimetry (ITC) and discovered that Aß42 and Aß40 bind to EVs in a manner that is sequence specific, saturable, and endothermic. In addition, Aß incubation with EVs overnight yielded larger amounts of bound Aß peptide that was fibrillar in structure. These findings point to a specific amyloid-EV interaction, a potential role for EVs in the transport of amyloid from the brain to the blood, and a role for this amyloid pool in the AD process.

Embedding of Precision-Cut Lung Slices in Engineered Hydrogel Biomaterials Supports Extended Ex Vivo Culture.

Maintaining the three-dimensional architecture and cellular complexity of lung tissue ex vivo can enable elucidation of the cellular and molecular pathways underlying chronic pulmonary diseases. Precision-cut lung slices (PCLS) are one human-lung model with the potential to support critical mechanistic studies and early drug discovery. However, many studies report short culture times of 7-10 days. Here, we systematically evaluated poly(ethylene glycol)-based hydrogel platforms for the encapsulation of PCLS. We demonstrated the ability to support ex vivo culture of embedded PCLS for at least 21 days compared with control PCLS floating in media. These customized hydrogels maintained PCLS architecture (no difference), viability (4.7-fold increase, P < 0.0001), and cellular phenotype as measured by SFTPC (1.8-fold increase, P < 0.0001) and vimentin expression (no change) compared with nonencapsulated controls. Collectively, these results demonstrate that hydrogel biomaterials support the extended culture times required to study chronic pulmonary diseases ex vivo using PCLS technology.

Clinical and Molecular Comparison of Pediatric and Adult Reverse Remodeling With Ventricular Assist Devices.

Ventricular assist device (VAD) support induces reverse remodeling of failing myocardium that leads to occasional functional recovery of the adult heart. While there have been numerous clinical reports in adult patients with end-stage cardiomyopathy, little is known about reverse remodeling in children, which has increasing clinical potential with the recent expansion of pediatric VADs in the setting of static organ supply for heart transplantation. Pediatric myocardium also promises theoretical advantages for recovery over adult myocardium due to its greater abundance of cardiac progenitor cells. To identify potential targets of future studies, we conducted a literature review with two aims: (i) to summarize clinical cases of pediatric patients who exhibited cardiac recovery following VAD support; and (ii) to analyze genetic changes in pediatric myocardium induced by VAD support compared with those observed in adult patients. Several clinical series of pediatric VAD cases report that small proportions of their cohorts were weaned off from device support, but a lack of information about the etiology and support duration of these patients limits the ability to determine whether they represent reverse remodeling of myocardial structure or just recovery from acute illness. A comparison of pediatric and adult gene expression changes with VAD support reveals approximately 40% of genes to be oppositely regulated, indicating that the pediatric genetic response is distinct. These observations highlight a necessity to better understand reverse remodeling specific to pediatric myocardium, which is crucial to improving clinical strategies for bridge-to-recovery in children.

Vascularization of PEGylated fibrin hydrogels increases the proliferation of human iPSC-cardiomyocytes.

Studies have long sought to develop engineered heart tissue for the surgical correction of structural heart defects, as well as other applications and vascularization of this tissue has presented a challenge. Recent studies suggest that vascular cells and a vascular network may have regenerative effects on implanted cardiomyocytes (CM) and nearby heart tissue separate from perfusion of oxygen and nutrients. The goal of this study was to test whether vascular cells or a formed vascular network in a fibrin-based hydrogel would alter the proliferation of human iPSC-derived CM. First, vascular network formation in a slowly degrading PEGylated fibrin hydrogel was optimized by altering the cell ratio of human umbilical vein endothelial cells to human dermal fibroblasts, the inclusion of growth factors, and the total cell concentration. An endothelial to fibroblast ratio of 5:1 and a total cell concentration of 1.1 × 106 cells/mL without additional growth factors generated robust vascular networks while minimizing the number of cells required. Using this optimized system, human iPSC-derived CM were cultured on hydrogels without vascular cells, hydrogels with unorganized encapsulated vascular cells, or hydrogels with encapsulated vascular cells organized into networks for 7 days. CM proliferation and gene expression were assayed following 7 days of culture on the hydrogels. The presence of vascular cells in the hydrogel, whether unorganized or in vascular networks, significantly increased CM proliferation compared to an acellular hydrogel. Hydrogels with unorganized vascular cells resulted in lower CM maturity evidenced by decreased expression of cardiac troponin t (TNNT2), myosin light chain 7, and phospholamban compared to hydrogels without vascular cells and hydrogels with vascular networks. Altogether, this study details a robust method of forming rudimentary vascular networks in a fibrin-based hydrogel and shows that a hydrogel containing endothelial cells and fibroblasts can induce proliferation in adjacent CM, and these cells do not hinder CM gene expression when organized into a vascular network.

Trisomy 21 Alters Cell Proliferation and Migration of iPSC-Derived Cardiomyocytes on Type VI Collagen.

Purpose: Individuals with Down syndrome (DS) are 2000 times more likely to develop a congenital heart defect (CHD) than the typical population Freeman et al. in Am J Med Genet 80:213-217 (1998). The majority of CHDs in individuals with DS characteristically involve the atrioventricular (AV) canal, including the valves and the atrial or ventricular septum. Type VI collagen (COLVI) is the primary structural component in the developing septa and endocardial cushions, with two of the three genes encoding for COLVI located on human chromosome 21 and upregulated in Down syndrome (von Kaisenberg et al. in Obstet Gynecol 91:319-323, 1998; Gittenberger-De Groot et al. in Anatom Rec Part A 275:1109-1116, 2023). Methods: To investigate the effect of COLVI dosage on cardiomyocytes with trisomy 21, induced pluripotent stem cells (iPSC) from individuals with DS and age- and sex-matched controls were differentiated into cardiomyocytes (iPSC-CM) and plated on varying concentrations of COLVI. Results: Real time quantitative PCR showed decreased expression of cardiac-specific genes of DS iPSC-CM lines compared to control iPSC-CM. As expected, DS iPSC-CM had increased expression of genes on chromosome 21, including COL6A1, COL6A2, as well as genes not located on chromosome 21, namely COL6A3, HAS2 and HYAL2. We found that higher concentrations of COLVI result in decreased proliferation and migration of DS iPSC-CM, but not control iPSC-CM. Conclusions: These results suggest that the increased expression of COLVI in DS may result in lower migration-driven elongation of endocardial cushions stemming from lower cell proliferation and migration, possibly contributing to the high incidence of CHD in the DS population. Supplementary Information: The online version contains supplementary material available at 10.1007/s12195-023-00791-x.

Formation of functional gap junctions in amniotic fluid-derived stem cells induced by transmembrane co-culture with neonatal rat cardiomyocytes.

Amniotic fluid-derived stem cells (AFSC) have been reported to differentiate into cardiomyocyte-like cells and form gap junctions when directly mixed and cultured with neonatal rat ventricular myocytes (NRVM). This study investigated whether or not culture of AFSC on the opposite side of a Transwell membrane from NRVM, allowing for contact and communication without confounding factors such as cell fusion, could direct cardiac differentiation and enhance gap junction formation. Results were compared to shared media (Transwell), conditioned media and monoculture media controls. After a 2-week culture period, AFSC did not express cardiac myosin heavy chain or troponin T in any co-culture group. Protein expression of cardiac calsequestrin 2 was up-regulated in direct transmembrane co-cultures and media control cultures compared to the other experimental groups, but all groups were up-regulated compared with undifferentiated AFSC cultures. Gap junction communication, assessed with a scrape-loading dye transfer assay, was significantly increased in direct transmembrane co-cultures compared to all other conditions. Gap junction communication corresponded with increased connexin 43 gene expression and decreased phosphorylation of connexin 43. Our results suggest that direct transmembrane co-culture does not induce cardiomyocyte differentiation of AFSC, though calsequestrin expression is increased. However, direct transmembrane co-culture does enhance connexin-43-mediated gap junction communication between AFSC.

Osmium tetroxide labeling of (poly)methyl methacrylate corrosion casts for enhancement of micro-CT microvascular imaging.

In order to enhance micro-computer tomography (micro-CT) imaging of corrosion casts of fine vasculature, metals can be added to the casting resin before perfusion. However, perfused metals lead to vasoconstriction or vessel damage resulting in nonphysiologic vascular casts. A novel method for coating methyl methacrylate vascular casts with osmium tetroxide has been developed in order to increase micro-CT contrast without affecting the vascular structure. This technique was verified using corrosion casts of the lung vasculature of New Zealand white rabbits. Osmium tetroxide coating of methyl methacrylate vascular corrosion casts resulted in an increase in overall sample contrast that translated into an increase in the resolution of the vasculature. This method can therefore lead to increased resolution in the characterization of fine vascular structures.

An In Vitro Characterization of a PCL-Fibrin Scaffold for Myocardial Repair.

Each year in the United States approximately 10,000 babies are born with a complex congenital heart defect (CHD) requiring surgery in the first year of after birth. Several of these operations require the implantation of a full-thickness heart patch; however, the current patch materials available to pediatric heart surgeons are exclusively non-living and non-degradable, which do not grow with the patient and are prone to fail due to an inability to integrate with the heart. In this work, the goal was to develop a full-thickness, tissue engineered myocardial patch (TEMP) that is made from biodegradable components, strong enough to withstand the mechanical forces of the heart wall, and able to integrate with the heart and drive neotissue formation. Here, a thick and porous electrospun PCL scaffold filled with high-salt PEGylated fibrin was developed. The scaffold was found to be mechanically sufficient for heart wall repair. Vascular cells were able to infiltrate more than halfway through the scaffold in static culture within three weeks. The scaffold maintained pluripotent stem cells for at least four days, supports viable iPSC-derived cardiomyocytes, and fostered tissue thickening in vitro. The TEMP developed here and tested in vitro is promising for the repair of structural CHD and will next be assessed in situ.

Bioreactor Design for Culturing Vascularized Engineered Tissue in Flow Conditions.

BACKGROUND: Current treatments for congenital heart defects often require surgery and implantation of a synthetic patch or baffle that becomes a fibrous scar and leads to a high number of reoperations. Previous studies in rats have shown that a pre-vascularized scaffold can integrate into the heart and result in regions of vascularized and muscularized tissue. However, increasing the thickness of this scaffold for use in human hearts requires a method to populate the thick scaffold and mature it under physiologic flow and electrical conditions. EXPERIMENT: We developed a bioreactor system that can perfuse up to six 7-mm porous scaffolds with tunable gravity-mediated flow and chronic electrical stimulation. Three polymers which have been reported to be biocompatible were evaluated for effects on the viability of induced pluripotent stem cell-derived cardiomyocytes (iPSC-CM). Bioreactor flow and electrical stimulation functions were tested, and the bioreactor was operated for up to 7 days to ensure reliability and lack of leaks in a 37C, humidified incubator. Height and flow relationships were measured for perfusion through an electrospun polycaprolactone (PCL) and gelatin scaffold previously reported by our laboratory. Culture with cells was evaluated by plating human umbilical vein endothelial cells (HUVEC) and human dermal fibroblasts (hDF) on top of the scaffolds in both static and flow conditions for 2,5 and 7 days. As a proof-of concept, scaffolds were cryosectioned and cell infiltration was quantified using immunofluorescence staining. RESULTS: Neither MED610 (Stratasys), Vero (Stratasys), nor FORMLAB materials affected the viability of iPSC derived cardiomyocytes, and MED610 was chosen for manufacture due to familiarity of 3D printing from this material. The generation of electrical field stimulation from 0 to 5 volts and physiological ranges of pump capacities were verified. The relationship between height and flow was calculated for scaffolds with and without cells. Finally, we demonstrated evaluation of cell depth and structure in scaffolds cultured for 2, 5, and 7 days. CONCLUSION: The gravity-mediated flow bioreactor system we developed can be used as a platform for 3D cell culture particularly designed for perfusing vascularized tissue constructs with electrical stimulation for cardiac maturation.

Chemokine-Induced PBMC and Subsequent MSC Migration Toward Decellularized Heart Valve Tissue.

PURPOSE: Enhancing the recellularization of a decellularized heart valve in situ may lead to an improved or ideal heart valve replacement. A promising approach is leveraging the immune response for inflammation-mediated recellularization. However, this mechanism has not been previously demonstrated in vitro. METHODS: This study investigated loading the chemokine MCP-1 into decellularized porcine heart valve tissue and measured the migration of human peripheral blood mononuclear cells (PBMCs) and mesenchymal stem cells (MSCs) toward the chemokine loaded valve tissue. RESULTS: The results of this study demonstrate that MCP-1-loaded tissues increase PBMC migration compared to non-loaded tissues. Additionally, we demonstrate MCP-1-loaded tissues that have recruited PBMCs lead to increased migration of MSCs compared to decellularized tissue alone. CONCLUSION: The results of this study provide evidence for the inflammation-mediated recellularization mechanism. Furthermore, the results support the use of such an approach for enhancing the recellularization of a decellularized heart valve.

A Prevascularized Polyurethane-Reinforced Fibrin Patch Improves Regenerative Remodeling in a Rat Right Ventricle Replacement Model.

Congenital heart defects (CHDs) affect 1 in 120 newborns in the United States. Surgical repair of structural heart defects often leads to arrhythmia and increased risk of heart failure. The laboratory has previously developed an acellular fibrin patch reinforced with a biodegradable poly(ether ester urethane) urea mesh that result in improved heart function when tested in a rat right ventricle wall replacement model compared to fixed pericardium. However, this patch does not drive significant neotissue formation. The patch materials are modified here and this patch is prevascularized with human umbilical vein endothelial cells and c-Kit+ human amniotic fluid stem cells. Rudimentary capillary-like networks form in the fibrin after culture of cell-encapsulated patches for 3 d in vitro. Prevascularized patches and noncell loaded patch controls are implanted onto full-thickness heart wall defects created in the right ventricle of athymic nude rats. Two months after surgery, defect repair with prevascularized patches results in improved heart function and the patched heart area exhibited greater vascularization and muscularization, less fibrosis, and increased M2 macrophage infiltration compared to acellular patches.

Increasing salinity of fibrinogen solvent generates stable fibrin hydrogels for cell delivery or tissue engineering.

Fibrin has been used clinically for wound coverings, surgical glues, and cell delivery because of its affordability, cytocompatibility, and ability to modulate angiogenesis and inflammation. However, its rapid degradation rate has limited its usefulness as a scaffold for 3D cell culture and tissue engineering. Previous studies have sought to slow the degradation rate of fibrin with the addition of proteolysis inhibitors or synthetic crosslinkers that require multiple functionalization or polymerization steps. These strategies are difficult to implement in vivo and introduce increased complexity, both of which hinder the use of fibrin in research and medicine. Previously, we demonstrated that additional crosslinking of fibrin gels using bifunctionalized poly(ethylene glycol)-n-hydroxysuccinimide (PEG-NHS) slows the degradation rate of fibrin. In this study, we aimed to further improve the longevity of these PEG-fibrin gels such that they could be used for tissue engineering in vitro or in situ without the need for proteolysis inhibitors. It is well documented that increasing the salinity of fibrin precursor solutions affects the resulting gel morphology. Here, we investigated whether this altered morphology influences the fibrin degradation rate. Increasing the final sodium chloride (NaCl) concentration from 145 mM (physiologic level) to 250 mM resulted in fine, transparent high-salt (HS) fibrin gels that degrade 2-3 times slower than coarse, opaque physiologic-salt (PS) fibrin gels both in vitro (when treated with proteases and when seeded with amniotic fluid stem cells) and in vivo (when injected subcutaneously into mice). Increased salt concentrations did not affect the viability of encapsulated cells, the ability of encapsulated endothelial cells to form rudimentary capillary networks, or the ability of the gels to maintain induced pluripotent stem cells. Finally, when implanted subcutaneously, PS gels degraded completely within one week while HS gels remained stable and maintained viability of seeded dermal fibroblasts. To our knowledge, this is the simplest method reported for the fabrication of fibrin gels with tunable degradation properties and will be useful for implementing fibrin gels in a wide range of research and clinical applications.

Assessment of electrospun cardiac patches made with sacrificial particles and polyurethane-polycaprolactone blends.

Congenital heart defects (CHDs) are the leading cause of death in live-born infants. Currently, patches used in the repair of CHDs are exclusively inert and non-degradable, which increases the risk of arrhythmia, follow-up surgeries, and sudden cardiac death. In this preliminary study, we sought to fabricate biodegradable scaffolds that can support cardiac regeneration in the repair of CHDs. We electrospun biodegradable scaffolds using various blends of polyurethane (PU) and polycaprolactone (PCL) with and without sacrificial poly(ethylene oxide) (PEO) particles and assessed the mechanical properties, cell infiltration levels, and inflammatory response in vitro (surface cell seeding) and in vivo (subcutaneous mouse implant). We hypothesized that a blend of the two polymers would preserve the low stiffness of PU as well as the high cell infiltration observed in PCL scaffolds. The inclusion of PU in the blends, even as low as 10%, decreased cell infiltration both in vitro and in vivo. The inclusion of sacrificial PEO increased pore sizes, reduced Young's moduli, and reduced the inflammatory response in all scaffold types. Collectively, we have concluded that a PCL patch electrospun with sacrificial PEO particles is the most promising scaffold for further assessment as in our heart defect model.

Review on Mechanical Support and Cell-Based Therapies for the Prevention and Recovery of the Failed Fontan-Kreutzer Circulation.

Though the current staged surgical strategy for palliation of single ventricle heart disease, culminating in a Fontan circulation, has increased short-term survival, mounting evidence has shown that the single ventricle, especially a morphologic right ventricle (RV), is inadequate for long-term circulatory support. In addition to high rates of ventricular failure, high central venous pressures (CVP) lead to liver fibrosis or cirrhosis, lymphatic dysfunction, kidney failure, and other comorbidities. In this review, we discuss the complications seen with Fontan physiology, including causes of ventricular and multi-organ failure. We then evaluate the clinical use, results, and limitations of long-term mechanical assist devices intended to reduce RV work and high CVP, as well as biological therapies for failed Fontan circulations. Finally, we discuss experimental tissue engineering solutions designed to prevent Fontan circulation failure and evaluate knowledge gaps and needed technology development to realize a more robust single ventricle therapy.

Evaluation of a polyurethane-reinforced hydrogel patch in a rat right ventricle wall replacement model.

Congenital heart defects affect about 1% births in the United States. Many of the defects are treated with surgically implanted patches made from inactive materials or fixed pericardium that do not grow with the patients, leading to an increased risk of arrhythmia, sudden cardiac death, and heart failure. This study investigated an angiogenic poly(ethylene glycol) fibrin-based hydrogel reinforced with an electrospun biodegradable poly(ether ester urethane) urea (BPUR) mesh layer that was designed to encourage cell invasion, angiogenesis, and regenerative remodeling in the repair of an artificial defect created onto the rat right ventricle wall. Electrocardiogram signals were analyzed, heart function was measured, and fibrosis, macrophage infiltration, muscularization, vascularization, and defect size were evaluated at 4- and 8-weeks post-surgery. Compared with rats with fixed pericardium patches, rats with BPUR-reinforced hydrogel patches had fewer arrhythmias and greater right ventricular ejection fraction and cardiac output, as well as greater left ventricular ejection fraction, fractional shorting, stroke work and cardiac output. Histology and immunofluorescence staining showed less fibrosis and less patch material remaining in rats with BPUR-reinforced hydrogel patches at 4- and 8-weeks. Rats with BPUR-reinforced hydrogel patches also had a greater volume of granular tissue, a greater volume of muscularized tissue, more blood vessels, and a greater number of leukocytes, pan-macrophages, and M2 macrophages at 8 weeks. Overall, this study demonstrated that the engineered BPUR-reinforced hydrogel patch initiated greater regenerative vascular and muscular remodeling with a limited fibrotic response, resulting in fewer incidences of arrhythmia and improved heart function compared with fixed pericardium patches when applied to heal the defects created on the rat right ventricle wall. STATEMENT OF SIGNIFICANCE: The study tested a polyurethane-reinforced hydrogel patch in a rat right ventricle wall replacement model. Compared with fixed pericardium patches, these reinforced hydrogel patches initiated greater regenerative vascular and muscular remodeling with a reduced fibrotic response, resulting in fewer incidences of arrhythmia and improved heart function at 4- and 8-weeks post surgery. Overall, the new BPUR-reinforced hydrogel patches resulted in better heart function when replacing contractile myocardium than fixed pericardium patches.

Engineering Myocardium for Heart Regeneration-Advancements, Considerations, and Future Directions.

Heart disease is the leading cause of death in the United States among both adults and infants. In adults, 5-year survival after a heart attack is <60%, and congenital heart defects are the top killer of liveborn infants. Problematically, the regenerative capacity of the heart is extremely limited, even in newborns. Furthermore, suitable donor hearts for transplant cannot meet the demand and require recipients to use immunosuppressants for life. Tissue engineered myocardium has the potential to replace dead or fibrotic heart tissue in adults and could also be used to permanently repair congenital heart defects in infants. In addition, engineering functional myocardium could facilitate the development of a whole bioartificial heart. Here, we review and compare in vitro and in situ myocardial tissue engineering strategies. In the context of this comparison, we consider three challenges that must be addressed in the engineering of myocardial tissue: recapitulation of myocardial architecture, vascularization of the tissue, and modulation of the immune system. In addition to reviewing and analyzing current progress, we recommend specific strategies for the generation of tissue engineered myocardial patches for heart regeneration and repair.

Epigenetics and Mechanobiology in Heart Development and Congenital Heart Disease.

: Congenital heart disease (CHD) is the most common birth defect worldwide and the number one killer of live-born infants in the United States. Heart development occurs early in embryogenesis and involves complex interactions between multiple cell populations, limiting the understanding and consequent treatment of CHD. Furthermore, genome sequencing has largely failed to predict or yield therapeutics for CHD. In addition to the underlying genome, epigenetics and mechanobiology both drive heart development. A growing body of evidence implicates the aberrant regulation of these two extra-genomic systems in the pathogenesis of CHD. In this review, we describe the stages of human heart development and the heart defects known to manifest at each stage. Next, we discuss the distinct and overlapping roles of epigenetics and mechanobiology in normal development and in the pathogenesis of CHD. Finally, we highlight recent advances in the identification of novel epigenetic biomarkers and environmental risk factors that may be useful for improved diagnosis and further elucidation of CHD etiology.

Substrate stiffness affects the functional maturation of neonatal rat ventricular myocytes.

Cardiac cells mature in the first postnatal week, concurrent with altered extracellular mechanical properties. To investigate the effects of extracellular stiffness on cardiomyocyte maturation, we plated neonatal rat ventricular myocytes for 7 days on collagen-coated polyacrylamide gels with varying elastic moduli. Cells on 10 kPa substrates developed aligned sarcomeres, whereas cells on stiffer substrates had unaligned sarcomeres and stress fibers, which are not observed in vivo. We found that cells generated greater mechanical force on gels with stiffness similar to the native myocardium, 10 kPa, than on stiffer or softer substrates. Cardiomyocytes on 10 kPa gels also had the largest calcium transients, sarcoplasmic calcium stores, and sarcoplasmic/endoplasmic reticular calcium ATPase2a expression, but no difference in contractile protein. We hypothesized that inhibition of stress fiber formation might allow myocyte maturation on stiffer substrates. Treatment of maturing cardiomyocytes with hydroxyfasudil, an inhibitor of RhoA kinase and stress fiber-formation, resulted in enhanced force generation on the stiffest gels. We conclude that extracellular stiffness near that of native myocardium significantly enhances neonatal rat ventricular myocytes maturation. Deviations from ideal stiffness result in lower expression of sarcoplasmic/endoplasmic reticular calcium ATPase, less stored calcium, smaller calcium transients, and lower force. On very stiff substrates, this adaptation seems to involve RhoA kinase.

Endothelial injury induces vascular smooth muscle cell proliferation in highly localized regions of a direct contact co-culture system.

Though previous studies have indicated a relationship between the proliferation of endothelial cells and vascular smooth muscle cells (VSMCs) in co-culture, the results have been contradictory and the signaling mechanism poorly understood. In this transmembrane co-culture study, VSMCs and endothelial cells were grown to confluence on opposite sides of a microporous membrane to mimic the intima/media border of vessels. The endothelial layer was injured, and then cultured for 3 days, resulting in partial re-endothelialization. VSMC proliferation across from the injured/partially recovered endothelial region was significantly higher than across from the de-endothelialized region (a sevenfold increase) and the uninjured region (a threefold increase). ELISA indicated that PDGF, which was undetectable in uninjured co-culture and homotypic controls, increased after injury and the addition of a piperazinyl-quinazoline carboxamide PDGF receptor inhibitor blocked VSMC proliferation across from the injured/partially recovered region. We conclude that co-culture signaling initiated by endothelial cell injury locally stimulates VSMC proliferation and that this signaling could be mediated by PDGF-BB.