Epicardial placement of human MSC-loaded fibrin sealant films for heart failure: Preclinical efficacy and mechanistic data.

Mesenchymal stromal cell (MSC) transplantation has been investigated as an advanced treatment of heart failure; however, further improvement of the therapeutic efficacy and mechanistic understanding are needed. Our previous study has reported that epicardial placement of fibrin sealant films incorporating rat amniotic membrane-derived (AM)-MSCs (MSC-dressings) could address limitations of traditional transplantation methods. To progress this finding toward clinical translation, this current study aimed to examine the efficacy of MSC-dressings using human AM-MSCs (hAM-MSCs) and the underpinning mechanism for myocardial repair. Echocardiography demonstrated that cardiac function and structure were improved in a rat ischemic cardiomyopathy model after hAM-MSC-dressing therapy. hAM-MSCs survived well in the rat heart, enhanced myocardial expression of reparative genes, and attenuated adverse remodeling. Copy number analysis by qPCR revealed that upregulated reparative genes originated from endogenous rat cells rather than hAM-MSCs. These results suggest hAM-MSC-dressing therapy stimulates a secondary release of paracrine factors from endogenous cells improving myocardial repair ("secondary paracrine effect"), and cardiac M2-like macrophages were identified as a potential cell source of repair. We demonstrated hAM-MSCs increased M2-like macrophages through not only enhancing M2 polarization but also augmenting their proliferation and migration capabilities via PGE2, CCL2, and TGF-ß1, resulting in enhanced cardiac function after injury.

Reparative macrophage transplantation for myocardial repair: a refinement of bone marrow mononuclear cell-based therapy.

Reparative macrophages play an important role in cardiac repair post-myocardial infarction (MI). Bone marrow mononuclear cells (BM-MNCs) have been investigated as a donor for cell therapy but with limited clinical success. These cells, however, may be utilized as a source for reparative macrophages. This translational study aimed to establish a robust in vitro protocol to produce functional reparative macrophages from BM-MNCs and to establish pre-clinical evidence of the efficacy of reparative macrophage transplantation for the treatment of MI. Mouse BM-MNCs were treated with M-CSF plus IL-4, IL-10, TGF-ß1 or combinations of these in vitro. The concomitant administration of M-CSF and IL-4 produced the highest rate and largest number of CD11b+F4/80+CD206+ reparative macrophages. Expression and secretion of tissue repair-related factors including IGF-1, TGF-ß1, VEGF and IL1-ra were remarkably enhanced in reparative macrophages compared to BM-MNCs. These cells were transplanted in a mouse MI model, resulting in evident improvement in cardiac function recovery, compared to BM-MNC transplantation. Histological studies showed that reparative macrophage transplantation enhanced myocardial tissue repair including augmented microvascular formation, reduced cardiomyocyte hypertrophy and attenuated interstitial fibrosis. Moreover, survival of reparative macrophages in the heart post-transplantation was increased compared to BM-MNCs. Reparative macrophage transplantation also increased host-derived reparative macrophages in part through TGF-ß secretion. In conclusion, concomitant M-CSF + IL-4 treatment effectively produced reparative macrophages from BM-MNCs in vitro. Transplantation of produced reparative macrophage achieved a superior therapeutic efficacy, compared to BM-MNC transplantation, through the enhanced quantity and quality of donor cell engraftment. Further development of this advanced cell-based therapy is warranted.

VEGF (Vascular Endothelial Growth Factor) Induces NRP1 (Neuropilin-1) Cleavage via ADAMs (a Disintegrin and Metalloproteinase) 9 and 10 to Generate Novel Carboxy-Terminal NRP1 Fragments That Regulate Angiogenic Signaling.

Objective- NRP1(neuropilin-1) acts as a coreceptor for VEGF (vascular endothelial growth factor) with an essential role in angiogenesis. Recent findings suggest that posttranslational proteolytic cleavage of VEGF receptors may be an important mechanism for regulating angiogenesis, but the role of NRP1 proteolysis and the NRP1 species generated by cleavage in endothelial cells is not known. Here, we characterize NRP1 proteolytic cleavage in endothelial cells, determine the mechanism, and investigate the role of NRP1 cleavage in regulation of endothelial cell function. Approach and Results- NRP1 species comprising the carboxy (C)-terminal and transmembrane NRP1 domains but lacking the ligand-binding A and B regions are constitutively expressed in endothelial cells. Generation of these C-terminal domain NRP1 proteins is upregulated by phorbol ester and Ca2+ ionophore, and reduced by pharmacological inhibition of metalloproteinases, by small interfering RNA-mediated knockdown of 2 members of ADAM (a disintegrin and metalloproteinase) family, ADAMs 9 and 10, and by a specific ADAM10 inhibitor. Furthermore, VEGF upregulates expression of these NRP1 species in an ADAM9/10-dependent manner. Transduction of endothelial cells with adenoviral constructs expressing NRP1 C-terminal domain fragments inhibited VEGF-induced phosphorylation of VEGFR2 (VEGF receptor tyrosine kinase)/KDR (kinase domain insert receptor) and decreased VEGF-stimulated endothelial cell motility and angiogenesis in coculture and aortic ring sprouting assays. Conclusions- These findings identify novel NRP1 species in endothelial cells and demonstrate that regulation of NRP1 proteolysis via ADAMs 9 and 10 is a new regulatory pathway able to modulate VEGF angiogenic signaling.

Cardiac Hypertrophy Is Inhibited by a Local Pool of cAMP Regulated by Phosphodiesterase 2.

RATIONALE: Chronic elevation of 3'-5'-cyclic adenosine monophosphate (cAMP) levels has been associated with cardiac remodeling and cardiac hypertrophy. However, enhancement of particular aspects of cAMP/protein kinase A signaling seems to be beneficial for the failing heart. cAMP is a pleiotropic second messenger with the ability to generate multiple functional outcomes in response to different extracellular stimuli with strict fidelity, a feature that relies on the spatial segregation of the cAMP pathway components in signaling microdomains. OBJECTIVE: How individual cAMP microdomains affect cardiac pathophysiology remains largely to be established. The cAMP-degrading enzymes phosphodiesterases (PDEs) play a key role in shaping local changes in cAMP. Here we investigated the effect of specific inhibition of selected PDEs on cardiac myocyte hypertrophic growth. METHODS AND RESULTS: Using pharmacological and genetic manipulation of PDE activity, we found that the rise in cAMP resulting from inhibition of PDE3 and PDE4 induces hypertrophy, whereas increasing cAMP levels via PDE2 inhibition is antihypertrophic. By real-time imaging of cAMP levels in intact myocytes and selective displacement of protein kinase A isoforms, we demonstrate that the antihypertrophic effect of PDE2 inhibition involves the generation of a local pool of cAMP and activation of a protein kinase A type II subset, leading to phosphorylation of the nuclear factor of activated T cells. CONCLUSIONS: Different cAMP pools have opposing effects on cardiac myocyte cell size. PDE2 emerges as a novel key regulator of cardiac hypertrophy in vitro and in vivo, and its inhibition may have therapeutic applications.

cGMP signals modulate cAMP levels in a compartment-specific manner to regulate catecholamine-dependent signaling in cardiac myocytes.

RATIONALE: cAMP and cGMP are intracellular second messengers involved in heart pathophysiology. cGMP can potentially affect cAMP signals via cGMP-regulated phosphodiesterases (PDEs). OBJECTIVE: To study the effect of cGMP signals on the local cAMP response to catecholamines in specific subcellular compartments. METHODS AND RESULTS: We used real-time FRET imaging of living rat ventriculocytes expressing targeted cAMP and cGMP biosensors to detect cyclic nucleotides levels in specific locales. We found that the compartmentalized, but not the global, cAMP response to isoproterenol is profoundly affected by cGMP signals. The effect of cGMP is to increase cAMP levels in the compartment where the protein kinase (PK)A-RI isoforms reside but to decrease cAMP in the compartment where the PKA-RII isoforms reside. These opposing effects are determined by the cGMP-regulated PDEs, namely PDE2 and PDE3, with the local activity of these PDEs being critically important. The cGMP-mediated modulation of cAMP also affects the phosphorylation of PKA targets and myocyte contractility. CONCLUSIONS: cGMP signals exert opposing effects on local cAMP levels via different PDEs the activity of which is exerted in spatially distinct subcellular domains. Inhibition of PDE2 selectively abolishes the negative effects of cGMP on cAMP and may have therapeutic potential.

Engineered cell-degradable poly(2-alkyl-2-oxazoline) hydrogel for epicardial placement of mesenchymal stem cells for myocardial repair.

Epicardial placement of mesenchymal stromal cells (MSCs) is a promising strategy for cardiac repair post-myocardial infarction, but requires the design of biomaterials to maximise the retention of donor cells on the heart surface and control their phenotype. To this end, we propose the use of a poly(2-alkyl-2-oxazoline) (POx) derivative, based on 2-ethyl-2-oxazoline and 2-butenyl-2-oxazoline. This POx polymer can be cured rapidly (less than 2 min) via photo-irradiation due to the use of di-cysteine cell degradable peptides. We report that the cell-degradable properties of the resulting POx hydrogels enables the regulation of cell protrusion in corresponding 3D matrices and that this, in turn, regulates the secretory phenotype of MSCs. In particular, the expression of pro-angiogenic genes was upregulated in partially cell-degradable POx hydrogels. Improved angiogenesis was confirmed in an in vitro microfluidic assay. Finally, we confirmed that, owing to the excellent tissue adhesive properties of thiol-ene crosslinked hydrogels, the epicardial placement of MSC-loaded POx hydrogels promoted the recovery of cardiac function and structure with reduced interstitial fibrosis and improved neovascular formation in a rat myocardial infarction model. This report demonstrates that engineered synthetic hydrogels displaying controlled mechanical, cell degradable and bioactive properties are particularly attractive candidates for the epicardial placement of stem cells to promote cardiac repair post myocardial infarction.

Cell barrier function of resident peritoneal macrophages in post-operative adhesions.

Post-operative adhesions are a leading cause of abdominal surgery-associated morbidity. Exposed fibrin clots on the damaged peritoneum, in which the mesothelial barrier is disrupted, readily adhere to surrounding tissues, resulting in adhesion formation. Here we show that resident F4/80HighCD206- peritoneal macrophages promptly accumulate on the lesion and form a 'macrophage barrier' to shield fibrin clots in place of the lost mesothelium in mice. Depletion of this macrophage subset or blockage of CD11b impairs the macrophage barrier and exacerbates adhesions. The macrophage barrier is usually insufficient to fully preclude the adhesion formation; however, it could be augmented by IL-4-based treatment or adoptive transfer of this macrophage subset, resulting in robust prevention of adhesions. By contrast, monocyte-derived recruited peritoneal macrophages are not involved in the macrophage barrier. These results highlight a previously unidentified cell barrier function of a specific macrophage subset, also proposing an innovative approach to prevent post-operative adhesions.

On-site fabrication of Bi-layered adhesive mesenchymal stromal cell-dressings for the treatment of heart failure.

Mesenchymal stromal/stem cell (MSC)-based therapy is a promising approach for the treatment of heart failure. However, current MSC-delivery methods result in poor donor cell engraftment, limiting the therapeutic efficacy. To address this issue, we introduce here a novel technique, epicardial placement of bi-layered, adhesive dressings incorporating MSCs (MSC-dressing), which can be easily fabricated from a fibrin sealant film and MSC suspension at the site of treatment. The inner layer of the MSC dressing, an MSC-fibrin complex, promptly and firmly adheres to the heart surface without sutures or extra glues. We revealed that fibrin improves the potential of integrated MSCs through amplifying their tissue-repair abilities and activating the Akt/PI3K self-protection pathway. Outer collagen-sheets protect the MSC-fibrin complex from abrasion by surrounding tissues and also facilitates easy handling. As such, the MSC-dressing technique not only improves initial retention and subsequent maintenance of donor MSCs but also augment MSC's reparative functions. As a result, this technique results in enhanced cardiac function recovery with improved myocardial tissue repair in a rat ischemic cardiomyopathy model, compared to the current method. Dose-dependent therapeutic effects by this therapy is also exhibited. This user-friendly, highly-effective bioengineering technique will contribute to future success of MSC-based therapy.

Fibrin Glue-aided, Instant Epicardial Placement Enhances the Efficacy of Mesenchymal Stromal Cell-Based Therapy for Heart Failure.

Transplantation of mesenchymal stromal cells (MSCs) is a promising new therapy for heart failure. However, the current cell delivery routes result in poor donor cell engraftment. We therefore explored the role of fibrin glue (FG)-aided, instant epicardial placement to enhance the efficacy of MSC-based therapy in a rat ischemic cardiomyopathy model. We identified a feasible and reproducible method to instantly produce a FG-MSC complex directly on the heart surface. This complex exhibited prompt, firm adhesion to the heart, markedly improving initial retention of donor MSCs compared to intramyocardial injection. In addition, maintenance of retained MSCs was enhanced using this method, together contributing the increased donor cell presence. Such increased donor cell quantity using the FG-aided technique led to further improved cardiac function in association with augmented histological myocardial repair, which correlated with upregulation of tissue repair-related genes. We identified that the epicardial layer was eliminated shortly after FG-aided epicardial placement of MSCs, facilitating permeation of the donor MSC's secretome into the myocardium enabling myocardial repair. These data indicate that FG-aided, on-site, instant epicardial placement enhances MSC engraftment, promoting the efficacy of MSC-based therapy for heart failure. Further development of this accessible, advanced MSC-therapy is justified.

IL-4 as a Repurposed Biological Drug for Myocardial Infarction through Augmentation of Reparative Cardiac Macrophages: Proof-of-Concept Data in Mice.

Recent research has shown that reparative (alternatively activated or M2) macrophages play a role in repair of damaged tissues, including the infarcted hearts. Administration of IL-4 is known to augment M2 macrophages. This translational study thus aimed to investigate whether IL-4 administration is useful for the treatment of myocardial infarction. Long-acting IL-4 complex (IL-4c; recombinant IL-4 mixed with anti-IL-4 monoclonal antibody as a stabilizer) was administered after coronary artery ligation in mice. It was observed that IL-4c administration increased accumulation of CD206+F4/80+ M2-like macrophages predominantly in the injured myocardium, compared to the control. Sorted cardiac M2-like macrophages highly expressed wide-ranging tissue repair-related genes. Indeed, IL-4c administration enhanced cardiac function in association with reduced infarct size and enhanced tissue repair (strengthened connective tissue formation, improved microvascular formation and attenuated cardiomyocyte hypertrophy). Experiments using Trib1 -/- mice that had a depleted ability to develop M2 macrophages and other in-vitro studies supported that these IL-4-mediated effects were induced via M2-like macrophages. On the other hand, when administered at Day 28 post-MI, the effects of IL-4c were diminished, suggesting a time-frame for IL-4 treatment to be effective. These data represent proof-of-concept of efficacy of IL-4 treatment for acute myocardial infarction, encouraging its further development.

Sustained exposure to catecholamines affects cAMP/PKA compartmentalised signalling in adult rat ventricular myocytes.

In the heart compartmentalisation of cAMP/protein kinase A (PKA) signalling is necessary to achieve a specific functional outcome in response to different hormonal stimuli. Chronic exposure to catecholamines is known to be detrimental to the heart and disrupted compartmentalisation of cAMP signalling has been associated to heart disease. However, in most cases it remains unclear whether altered local cAMP signalling is an adaptive response, a consequence of the disease or whether it contributes to the pathogenetic process. We have previously demonstrated that isoforms of PKA expressed in cardiac myocytes, PKA-I and PKA-II, localise to different subcellular compartments and are selectively activated by spatially confined pools of cAMP, resulting in phosphorylation of distinct downstream targets. Here we investigate cAMP signalling in an in vitro model of hypertrophy in primary adult rat ventricular myocytes. By using a real time imaging approach and targeted reporters we find that that sustained exposure to catecholamines can directly affect cAMP/PKA compartmentalisation. This appears to involve a complex mechanism including both changes in the subcellular localisation of individual phosphodiesterase (PDE) isoforms as well as the relocalisation of PKA isoforms. As a result, the preferential coupling of PKA subsets with different PDEs is altered resulting in a significant difference in the level of cAMP the kinase is exposed to, with potential impact on phosphorylation of downstream targets.

Neuropilins 1 and 2 mediate neointimal hyperplasia and re-endothelialization following arterial injury.

AIMS: Neuropilins 1 and 2 (NRP1 and NRP2) play crucial roles in endothelial cell migration contributing to angiogenesis and vascular development. Both NRPs are also expressed by cultured vascular smooth muscle cells (VSMCs) and are implicated in VSMC migration stimulated by PDGF-BB, but it is unknown whether NRPs are relevant for VSMC function in vivo. We investigated the role of NRPs in the rat carotid balloon injury model, in which endothelial denudation and arterial stretch induce neointimal hyperplasia involving VSMC migration and proliferation. METHODS AND RESULTS: NRP1 and NRP2 mRNAs and proteins increased significantly following arterial injury, and immunofluorescent staining revealed neointimal NRP expression. Down-regulation of NRP1 and NRP2 using shRNA significantly reduced neointimal hyperplasia following injury. Furthermore, inhibition of NRP1 by adenovirally overexpressing a loss-of-function NRP1 mutant lacking the cytoplasmic domain (ΔC) reduced neointimal hyperplasia, whereas wild-type (WT) NRP1 had no effect. NRP-targeted shRNAs impaired, while overexpression of NRP1 WT and NRP1 ΔC enhanced, arterial re-endothelialization 14 days after injury. Knockdown of either NRP1 or NRP2 inhibited PDGF-BB-induced rat VSMC migration, whereas knockdown of NRP2, but not NRP1, reduced proliferation of cultured rat VSMC and neointimal VSMC in vivo. NRP knockdown also reduced the phosphorylation of PDGFα and PDGFß receptors in rat VSMC, which mediate VSMC migration and proliferation. CONCLUSION: NRP1 and NRP2 play important roles in the regulation of neointimal hyperplasia in vivo by modulating VSMC migration (via NRP1 and NRP2) and proliferation (via NRP2), independently of the role of NRPs in re-endothelialization.

The relationship between clinical measurements of lower extremity posture and tibial translation.

OBJECTIVE: The purpose of this investigation was to determine if postures of the lower extremity were related to the amount of anterior tibial translation. DESIGN: Regression model of lower extremity postural measures used to predict the amount of tibial translation for a sample of convenience. BACKGROUND: Retrospective studies have indicated a link between certain lower extremity postures and prediction of anterior cruciate ligament injury status. What is not clear is whether these lower extremity postures cause, or occur as a result of anterior cruciate ligament injury. METHODS: Genu recurvatum, the thigh-foot angle, and navicular drop measures were obtained from the right leg of 43 uninjured college-aged subjects. Tibial translation was determined using a KT-1000 arthrometer. Gender related differences were determined with independent t-tests. Step-wise regression was performed to assess the relationship between postural measures and subject gender, and tibial translation. RESULTS: Very good to excellent inter- and intra-rater reliability was found for the postural measures. There was greater tibial translation (P=0.01) in females. The final regression model indicated a moderate relationship between sex of the subject, navicular drop and tibial translation (r=0.551). Genu recurvatum and thigh-foot angle were not significant predictors of tibial translation. CONCLUSIONS; We concluded that foot pronation has a greater impact on the amount of tibial translation than genu recurvatum and torsion of the lower leg in anterior cruciate intact subjects. RELEVANCE: Static clinical measures of lower extremity posture are commonly taken as predictors of outcome (both performance and injury). There may be little association between these static measures and dynamic performance.

Analysis of compartmentalized cAMP: a method to compare signals from differently targeted FRET reporters.

Förster resonance energy transfer (FRET)-based reporters are important tools to study the spatiotemporal compartmentalization of cyclic adenosine monophosphate (cAMP) in living cells. To increase the spatial resolution of cAMP detection, new reporters with specific intracellular targeting have been developed. Therefore it has become critical to be able to appropriately compare the signals revealed by the different sensors. Here we illustrate a protocol to calibrate the response detected by different targeted FRET reporters involving the generation of a dose-response curve to the cAMP raising agent forskolin. This method represents a general tool for the accurate analysis and interpretation of intracellular cAMP changes detected at the level of different subcellular compartments.

Renal and vascular glutathione S-transferase mu is not affected by pharmacological intervention to reduce systolic blood pressure.

BACKGROUND: Our previous studies demonstrated reduced rat glutathione S-transferase mu type 1 (Gstm1) expression in stroke-prone spontaneously hypertensive rats (SHRSPs), when compared with the normotensive Wistar-Kyoto rat. METHODS: This study investigated the effects of angiotensin II type 1 receptor blocker (ARB) and a diuretic/vasodilator combination on the expression levels of rat Gstm1 and other Gstm isoforms. RESULTS: Antihypertensive treatments of young and mature SHRSPs with an ARB and a diuretic/vasodilator combination improved SBP but did not affect the expression levels of Gstm1. Although Gstm1 is a member of a family of highly homologous genes, with the exception of Gstm2, there was no evidence for compensatory increase in expression of other Gstm isoforms. In contrast, we observed reduced expression of several other Gstm isoforms in untreated SHRSPs. Untreated SHRSPs demonstrated increased renal and vascular oxidative stress, both of which were not significantly affected by the antihypertensive treatments. Untreated SHRSPs scored significantly higher when assessed for renal histopathological damage, and this was improved by antihypertensive treatments. CONCLUSION: These results suggest that reduced Gstm1 expression in SHRSPs is due to strain-dependent genetic abnormalities, playing a causative role in the development of hypertension, probably through oxidative stress pathway. Renal changes occur as a consequence of increased blood pressure and can be improved when treated with antihypertensive drugs. In silico comparative genome analysis combined with expression studies in rat and human vascular tissue revealed that there are possible four human homologues (GSTM1, GSTM2, GSTM4 and GSTM5) for rat Gstm1.