JNK2 Gene Silencing for Elastic Matrix Regenerative Repair.

Elastic fibers do not naturally regenerate in many proteolytic disorders, such as in abdominal aortic aneurysms, and prevent restoration of tissue homeostasis. We have shown drug-based attenuation of the stress-activated protein kinase, JNK-2 to stimulate elastic matrix neoassembly and to attenuate cellular proteolytic activity. We now investigate if JNK2 gene knockdown with small interfering RNA (siRNA) provides greater specificity of action and improved regenerative/antiproteolytic outcomes in a proteolytic injury culture model of rat aneurysmal smooth muscle cells (EaRASMCs). A siRNA dose of 12.5 nM delivered with a transfection reagent significantly enhanced downstream elastic fiber assembly and maturation versus untreated EaRASMC cultures. The optimal siRNA dose was also delivered as a complex with a polymeric transfection vector, polyethyleneimine (PEI) in preparation for future in vivo delivery. Linear 25 kDa PEI-siRNA (5:1 molar ratio of amine to phosphate) and linear 40 kDa PEI-siRNA (2.5:1 ratio) were effective in downregulating the JNK2 gene, and significantly increasing expression of elastic fiber assembly proteins, and decreases in elastolytic matrix metalloprotease-2 versus treatment controls to significantly increase mature elastic fiber assembly. The current work has identified siRNA dosing and siRNA-PEI complexing conditions that are safe and efficient in stimulating processes contributing to improved elastic matrix neoassembly via JNK2 gene knockdown. The results represent a mechanistic basis of a broader therapeutic approach to reverse elastic matrix pathophysiology in tissue disorders involving aberrations of elastic matrix homeostasis, such as in aortic aneurysms. Impact statement The elastic matrix and elastic fibers are key components of the structural extracellular matrix of elastic tissues and are essential to their stretch and recoil and to maintain healthy cell phenotype. Regeneration and repair of elastic matrix is naturally poor and impaired and is an unresolved challenge in tissue engineering. In this work, we investigate a novel gene silencing approach based on inhibiting the JNK2 gene, which provides significant downstream benefits to elastic fiber assembly and maturation. Combined with novel delivery strategies such as nanoparticles, we expect our approach to effect in situ elastic matrix repair in the future.

Pro-elastogenic effects of mesenchymal stem cell derived smooth muscle cells in a 3D collagenous milieu.

Intrinsically poor auto-regenerative repair of proteolytically-disrupted elastic matrix structures by resident SMCs in the wall of abdominal aortic aneurysms (AAAs) prevents growth arrest and regression of these wall expansions. Supporting their possible future use in a regenerative cell therapy for AAAs, in a prior study, we showed that bone marrow mesenchymal stem cell-derived Smooth Muscle Cells (BM-SMCs) secrete biological factors that have significant pro-elastogenic and anti-proteolytic effects on aneurysmal rat aortic SMCs (EaRASMCs) in non-contact co-cultures. We also identified one stable BM-SMC phenotype (cBM-SMC) generated by differentiating BM-MSCs on a 2D fibronectin substrate in the presence of PDGF (Platelet Derived Growth Factor) and TGF-ß1 (Transforming Growth Factor-ß1) that exhibited superior elastogenicity and pro-elastogenic/anti-proteolytic properties. In this study, we further investigated the ability of these cBM-SMCs to maintain these superior elastogenic properties in a 3D collagenous milieu alone and in co-culture with EaRASMC to evaluate their potential as an alternative cell source for cell therapy in AAA. Some of our key observations were higher contractility and greater amount of structurally intact elastin production in both standalone culture of cBM-SMCs as well as co-culture of cBM-SMCs with EaRASMCs as shown by VVG (Verhoeff-Van Gieson) staining and Pontamine Sky Blue labeling and lower MMP-9 protein expression in standalone culture in 3D collagenous environment. Our overall result indicates that cBM-SMCs possess the ability to provide elastogenic impetus in a 3D collagenous AAA milieu which is otherwise not conducive to elastogenesis. Therefore our study strongly suggest the utility of cBM-SMCs as a potential cell source for cell therapy to augment elastic matrix neo-assembly and fiber formation and attenuate proteolysis in a collagenous milieu that is evocative of the de-elasticized aneurysmal wall. STATEMENT OF SIGNIFICANCE: Abdominal aortic aneurysm (AAA) or ballooning of the aorta is one of the leading causes of cardiovascular disease (CVD) related death caused by significantly increased proteolytic activity in the aortic wall. Reversing pathophysiology of this condition is challenging due to intrinsically poor regeneration of elastin by aortic smooth muscle cells. Current management of AAA is limited to passive monitoring of the disease until it becomes large enough to receive surgical intervention and no drug based therapy currently exists. Cell based therapy can be a potential alternative treatment in this scenario because it provides elastogenic impetus to the aneurysmal SMCs, compensates for the dead SMCs and serves as a robust source of elastin while being delivered with minimal invasiveness. Hence this work will have significant impact in the field of tissue engineering and regenerative medicine.

MAGP-1 and fibronectin control EGFL7 functions by driving its deposition into distinct endothelial extracellular matrix locations.

The extracellular matrix (ECM) is essential to provide mechanical support to tissues but is also a bioactive edifice which controls cell behavior. Cell signaling generated by ECM components through integrin-mediated contacts, modulates cell biological activity. In addition, by sequestrating or releasing growth factors, the ECM is an active player of physiological and pathological processes such as vascular development. EGFL7 is mainly expressed during blood vessel development and is deposited in the ECM after secretion by endothelial cells. While EGFL7 is known to control various endothelial cell molecular mechanisms [i.e., the repression of endothelial-derived lysyl oxidase (LOX) enzyme, the regulation of the Notch pathway, and the expression of leukocyte adhesion molecules and of RHOA by endothelial cells], it is not established whether EGFL7 functions when bound to the ECM. Here, we show that microfibrillar-associated glycoprotein-1 (MAGP-1) and fibronectin drive the deposition of EGFL7 into both fibers and individual aggregates in endothelial ECM. Although EGFL7 does not need to be docked into the ECM to control endothelial adhesion molecule expression, the ECM accumulation of EGFL7 is required for its regulation of LOX activity and of HEY2 expression along the Notch pathway. The interaction of EGFL7 with MAGP-1 is necessary for LOX activity repression by EGFL7 while it does not participate in the control of the Notch pathway by this protein. Altogether, this study highlights the roles played by EGFL7 in controlling various endothelial molecular mechanisms upon its localization and shows how the ECM can modulate its functions.

Phenotype-based selection of bone marrow mesenchymal stem cell-derived smooth muscle cells for elastic matrix regenerative repair in abdominal aortic aneurysms.

Chronic proteolytic disruption of elastic fibres within the abdominal aortic wall results in wall vessel expansion to form rupture-prone abdominal aortic aneurysms (AAA). Arresting AAA growth is not possible as adult vascular smooth muscle cells (SMCs) poorly auto-regenerate and repair elastic fibres. Thus, there is a need to identify alternate cell sources capable of robust elastic matrix assembly to overcome elastolysis in the AAA wall. Previously, we demonstrated the superior elastogenic properties of rat bone marrow mesenchymal stem cell (BM-MSC)-derived SMCs (BM-SMCs) relative to aneurysmal and healthy rat aortic SMCs. In the present study, we investigate how phenotypic coordinates of the derived BM-SMCs, in turn dependent on conditions of BM-MSC differentiation, impact their elastic matrix synthesis abilities. More specifically, we investigated how glucose content, serum levels and the presence of transforming growth factor (TGF)-ß1 supplements alone or together with platelet-derived growth factor (PDGF-BB) in the differentiation medium influence phenotype of, and elastogenesis by derived rat BM-SMCs. BM-SMCs generated in low-glucose and 10% v/v serum conditions in the presence of TGF-ß1 with or without PDGF-BB exhibited a mature phenotype characterized by contractility and migrative tendencies similar to healthy rat aortic SMCs, and yet capable of robust tropoelastin (precursor) synthesis and assembly of a fibrous, highly crosslinked elastic matrix. Thus, we have identified metrics and conditions for selecting BM-SMCs with superior elastogenesis for in situ elastic matrix regeneration. Future studies will focus on characterizing these specific BM-SMC subtypes for their pro-elastogenic and anti-proteolytic effects on aneurysmal SMCs to confirm their preferred use for therapy aimed at AAA tissue regenerative repair. Copyright © 2016 John Wiley & Sons, Ltd.

Multifunctional, JNK-inhibiting nanotherapeutics for augmented elastic matrix regenerative repair in aortic aneurysms.

Growth of abdominal aortic aneurysms (AAA), localized aortal wall expansions, is driven by the disruption and subsequent loss of aortal wall elastic fibers by matrix metalloproteases (MMPs). Since elastic fibers do not naturally regenerate or repair, arresting/reversing AAA growth has not been possible. Previously, we showed utility of doxycycline (DOX), an MMP inhibitor drug, to stimulate elastic matrix neoassembly and crosslinking at low microgram per milliliter doses in addition to inhibiting MMPs. We currently show in aneurysmal smooth muscle cell (SMC) cultures that effects of exogenous DOX in this dose range are linked to its upregulation of transforming growth factor beta (TGF-ß1) via its inhibition of the regulatory protein c-Jun-N-terminal kinase 2 (JNK 2). We have identified a DOX dose range that stimulates elastogenesis and crosslinking without adversely impacting cell viability. Using JNK 2 inhibition as a metric for pro-regenerative matrix effects of DOX, we further demonstrate that sustained, steady-state release of DOX at the useful dose, from poly(ethylene glycol)-poly(lactic glycolic acid) nanoparticles (NPs), provides pro-elastogenic and anti-proteolytic effects that could potentially be more pronounced than that of exogenous DOX. We attribute these outcomes to previously determined synergistic effects provided by cationic amphiphile groups functionalizing the polymer NP surface. Released DOX inhibited expression and phosphorylation of JNK to likely increase expression of TGF-ß1, which is known to increase elastogenesis and lysyl oxidase-mediated crosslinking of elastic matrix. Our results suggest that JNK inhibition is a useful metric to assess pro-elastic matrix regenerative effects and point to the combinatorial regenerative benefits provided by DOX and cationic-functionalized NPs.

Comparative biology of decellularized lung matrix: Implications of species mismatch in regenerative medicine.

Lung engineering is a promising technology, relying on re-seeding of either human or xenographic decellularized matrices with patient-derived pulmonary cells. Little is known about the species-specificity of decellularization in various models of lung regeneration, or if species dependent cell-matrix interactions exist within these systems. Therefore decellularized scaffolds were produced from rat, pig, primate and human lungs, and assessed by measuring residual DNA, mechanical properties, and key matrix proteins (collagen, elastin, glycosaminoglycans). To study intrinsic matrix biologic cues, human endothelial cells were seeded onto acellular slices and analyzed for markers of cell health and inflammation. Despite similar levels of collagen after decellularization, human and primate lungs were stiffer, contained more elastin, and retained fewer glycosaminoglycans than pig or rat lung scaffolds. Human endothelial cells seeded onto human and primate lung tissue demonstrated less expression of vascular cell adhesion molecule and activation of nuclear factor-κB compared to those seeded onto rodent or porcine tissue. Adhesion of endothelial cells was markedly enhanced on human and primate tissues. Our work suggests that species-dependent biologic cues intrinsic to lung extracellular matrix could have profound effects on attempts at lung regeneration.

Sterilization of Lung Matrices by Supercritical Carbon Dioxide.

Lung engineering is a potential alternative to transplantation for patients with end-stage pulmonary failure. Two challenges critical to the successful development of an engineered lung developed from a decellularized scaffold include (i) the suppression of resident infectious bioburden in the lung matrix, and (ii) the ability to sterilize decellularized tissues while preserving the essential biological and mechanical features intact. To date, the majority of lungs are sterilized using high concentrations of peracetic acid (PAA) resulting in extracellular matrix (ECM) depletion. These mechanically altered tissues have little to no storage potential. In this study, we report a sterilizing technique using supercritical carbon dioxide (ScCO2) that can achieve a sterility assurance level 10(-6) in decellularized lung matrix. The effects of ScCO2 treatment on the histological, mechanical, and biochemical properties of the sterile decellularized lung were evaluated and compared with those of freshly decellularized lung matrix and with PAA-treated acellular lung. Exposure of the decellularized tissue to ScCO2 did not significantly alter tissue architecture, ECM content or organization (glycosaminoglycans, elastin, collagen, and laminin), observations of cell engraftment, or mechanical integrity of the tissue. Furthermore, these attributes of lung matrix did not change after 6 months in sterile buffer following sterilization with ScCO2, indicating that ScCO2 produces a matrix that is stable during storage. The current study's results indicate that ScCO2 can be used to sterilize acellular lung tissue while simultaneously preserving key biological components required for the function of the scaffold for regenerative medicine purposes.

Novel chemo-sensitizing agent, ERW1227B, impairs cellular motility and enhances cell death in glioblastomas.

Glioblastomas display variable phenotypes that include increased drug-resistance associated with enhanced migratory and anti-apoptotic characteristics. These shared characteristics contribute to failure of clinical treatment regimens. Identification of novel compounds that promote cell death and impair cellular motility is a logical strategy to develop more effective clinical protocols. We recently described the ability of the small molecule, KCC009, a tissue transglutaminase (TG2) inhibitor, to sensitize glioblastoma cells to chemotherapy. In the current study, we synthesized a series of related compounds that show variable ability to promote cell death and impair motility in glioblastomas, irrespective of their ability to inhibit TG2. Each compound has a 3-bromo-4,5-dihydroisoxazole component that presumably reacts with nucleophilic cysteine thiol residues in the active sites of proteins that have an affinity to the small molecule. Our studies focused on the effects of the compound, ERW1227B. Treatment of glioblastoma cells with ERW1227B was associated with both down-regulation of the PI-3 kinase/Akt pathway, which enhanced cell death; as well as disruption of focal adhesive complexes and intracellular actin fibers, which impaired cellular mobility. Bioassays as well as time-lapse photography of glioblastoma cells treated with ERW1227B showed cell death and rapid loss of cellular motility. Mice studies with in vivo glioblastoma models demonstrated the ability of ERW1227B to sensitize tumor cells to cell death after treatment with either chemotherapy or radiation. The above findings identify ERW1227B as a potential novel therapeutic agent in the treatment of glioblastomas.