Engineered resveratrol-loaded fibrous scaffolds promotes functional cardiac repair and regeneration through Thioredoxin-1 mediated VEGF pathway.

Despite recent advancements, mortality due to coronary heart disease (CHD) remains high. Recently, the use of tissue-engineered grafts and scaffolds has emerged as a candidate for supporting the myocardium after an ischemic event. Resveratrol is a naturally occurring plant-based non-flavonoid polyphenolic compound found in many natural foods, including grapes and red wine. We embedded resveratrol in a polycaprolactone (PCL) scaffold and evaluated the cardio-therapeutic effects in a murine model of myocardial infarction (MI), with animals being grouped into Sham (S), Myocardial Infarction (MI), MI + PCL, and MI + PCL-Resveratrol (MI + PCL-R). After 4 and 8 weeks, echocardiography was performed to assess ejection fraction (EF) and fractional shortening (FS), which was followed by immunohistochemistry and immunofluorescence analysis at 8 weeks. The MI + PCL-R group showed a significant improvement in EF and FS compared with the MI + PCL group at 4 and 8-weeks post-surgery. PCL-R scaffolds treated hearts revealed decreased inflammatory cell infiltration, improved collagen extracellular matrix (ECM) secretion and blood vessel network formation following MI. The immunofluorescence analysis revealed resveratrol-loaded scaffolds promote increased expression of cTnT, Cx-43, Trx-1, and VEGF proteins. This study reports resveratrol-mediated rescue of ischemic myocardium when delivered through a biodegradable polymeric scaffold system after MI.

Evaluation of dermal tissue regeneration using resveratrol loaded fibrous matrix in a preclinical mouse model of full-thickness ischemic wound.

The complete loss of dermal tissue due to ischemia is a serious challenge facing clinicians. Frequently, the failure of wound healing is due to ischemic conditions prevailing at the site of damaged tissue. Restoration of lost vasculature at the ischemic site can be achieved by supplementing proangiogenic stimuli through an engineered scaffold mimicking dermal extracellular matrix. Towards this objective, we have developed an electrospun scaffold loaded with the pro-angiogenic molecule resveratrol. The physical and chemical changes in the polymeric scaffold before and after loading of resveratrol were characterized using field emission scanning electron microscopy (FE-SEM), Fourier-transform infrared spectroscopy (FT-IR), differential scanning calorimetry (DSC), thermogravimetric analysis (TGA), coherence scanning interferometry (CSI) and X-ray diffraction (XRD). A sustained release of resveratrol from the scaffold was elucidated by UV-spectrophotometer analysis. The enhancement in cell-matrix interaction was studied using human umbilical vein endothelial cells (HUVECs) seeded on the scaffolds. The biocompatibility analysis of resveratrol loaded scaffolds was evaluated through a subcutaneous implantation study in mice. The therapeutic potential of resveratrol loaded scaffolds to accelerate tissue repair was analyzed in a full-thickness ischemic wound model in mice. Wound closure and H&E staining analysis showed rapid closure of ischemic wound area and re-epithelialization in resveratrol loaded scaffold treated groups compared to collagen and negative control groups. The immunostaining analysis further revealed the activation of thioredoxin-1 (Trx-1), heme oxygenase-1 (HO-1) mediated vascular endothelial growth factor (VEGF) signaling in resveratrol loaded scaffold treated group. The expression of Bcl-2 in healing wound edges post-treatment with resveratrol loaded scaffold confirmed the anti-apoptotic effect mediated by resveratrol. From this study, we explored a synergistic effect mediated by resveratrol and fibrous scaffolds to aid the ischemic wound healing process through effective vascularization.

Thioredoxin-1 augments wound healing and promote angiogenesis in a murine ischemic full-thickness wound model.

BACKGROUND: Nonhealing wounds are a continuing health problem in the United States. Overproduction of reactive oxygen species is a major causative factor behind delayed wound healing. Previously we reported that thioredoxin-1 treatment could alleviate oxidative stress under ischemic conditions, such as myocardial infarction and hindlimb ischemia. In this study, we explored the potential for thioredoxin-1 gene therapy to effectively aid wound healing through improved angiogenesis in a murine ischemic wound model. METHODS: Full-thickness, cutaneous, ischemic wounds were created in the dorsum skin flap of 8- to 12-week-old CD1 mice. Nonischemic wounds created lateral to the ischemic skin flap served as internal controls. Mice with both ischemic wounds and nonischemic wounds were treated with Adeno-LacZ (1 × 109 pfu) or Adeno-thioredoxin-1 (1 × 109 pfu), injected intradermally around the wound. Digital imaging was performed on days 0, 3, 6, and 9 to assess the rate of wound closure. Tissue samples collected at predetermined time intervals were processed for immunohistochemical analysis. RESULTS: No significant differences in wound closure were identified among the nonischemic wounds control, nonischemic wounds-LacZ, and nonischemic wounds-thioredoxin-1 groups. Hence, only mice with ischemic wounds were further analyzed. The ischemic wounds-thioredoxin-1 group had significant improvement in wound closure on days 6 and 9 after surgery compared with the ischemic wounds control and ischemic wounds-LacZ groups. Immunohistochemical analysis indicated increased thioredoxin-1, vascular endothelial cell growth factor, and ß-catenin levels in the ischemic wounds-thioredoxin-1 group compared with the ischemic wounds control and ischemic wounds-LacZ groups, as well as increased capillary density and cell proliferation, as represented by Ki-67 staining. CONCLUSION: Taken together, thioredoxin-1 gene therapy promotes vascular endothelial cell growth factor signaling and re-epithelialization and activates wound closure in mice with ischemic wounds.

Graphene-based drug delivery systems in tissue engineering and nanomedicine.

The time and dosage form of graphene derivatives have been found to determine therapeutic and toxic windows in several cell lines and preclinical models. The enhanced biological action of graphene derivatives is made possible by altering the chemistry of native materials via surface conjugation, or by changing the oxidation state. The high level of chemical reactivity vested in the planar structure of graphene can be used to load various drugs and biomolecules with maximum radical scavenging effect. The integration of graphene and polymers brings electrical conductivity to scaffolds, making them ideal for cardiac or neuronal tissue engineering. Drawbacks associated with graphene-based materials for biomedical applications include defect-free graphene formation and heteroatom contamination during synthesis process; reduced availability of sp2 hybridized carbon centers due to serum proteins masking; and poor availability of data pertaining to in vivo clearance of graphene-based formulations. Personalized medicine is an emerging area of alternative treatments, which in combination with graphene-based nanobiomaterials, has revolutionary potential for the development of individualized nanocarriers to treat highly challenging diseases.

Development of next generation cardiovascular therapeutics through bio-assisted nanotechnology.

Cardiovascular diseases (CVDs) rank, next to cancer and stroke, among the deadliest diseases in the world. Among the major CVDs, acute myocardial infarction is a life-threatening disorder resulting from permanent damage to the left ventricular cardiac tissue. The major coronary arteries that supply blood to the functional left ventricle become blocked due to thrombotic plaque occlusion. During myocardial ischemia, oxidative stress and free radicals destroy healthy cardiomyocytes, smooth muscle cells, and endothelial cells, followed by degradation of the extracellular matrix, which results in ventricular wall-thinning and dilation. To protect the left ventricle from further damage and to rescue ischemic cardiac tissue, specially designed scaffolds consisting of biological material and nanomaterials have been developed. At the preclinical level, scaffolds loaded with growth factors and cells have been shown to regenerate ischemic tissue into healthy, functional myocardium. In this review, different therapeutic strategies currently available to treat the disease conditions at various stages are discussed, with special emphasis on biomaterials. Recent advancements in cardiovascular therapeutics using graphene and exosomal nanovesicles are discussed in detail. In addition, future directions for the development of next-generation cardiovascular therapeutics with biological and non-biological materials through nano-assisted technology are explored. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 2072-2083, 2018.

Thioredoxin-1 attenuates sepsis-induced cardiomyopathy after cecal ligation and puncture in mice.

BACKGROUND: Sepsis is a leading cause of mortality among patients in intensive care units across the USA. Thioredoxin-1 (Trx-1) is an essential 12 kDa cytosolic protein that, apart from maintaining the cellular redox state, possesses multifunctional properties. In this study, we explored the possibility of controlling adverse myocardial depression by overexpression of Trx-1 in a mouse model of severe sepsis. METHODS: Adult C57BL/6J and Trx-1Tg/+ mice were divided into wild-type sham (WTS), wild-type cecal ligation and puncture (WTCLP), Trx-1Tg/+sham (Trx-1Tg/+S), and Trx-1Tg/+CLP groups. Cardiac function was evaluated before surgery, 6 and 24 hours after CLP surgery. Immunohistochemical and Western blot analysis were performed after 24 hours in heart tissue sections. RESULTS: Echocardiography analysis showed preserved cardiac function in the Trx-1Tg/+ CLP group compared with the WTCLP group. Similarly, Western blot analysis revealed increased expression of Trx-1, heme oxygenase-1 (HO-1), survivin (an inhibitor of apoptosis [IAP] protein family), and decreased expression of thioredoxin-interacting protein (TXNIP), caspase-3, and 3- nitrotyrosine in the Trx-1Tg/+CLP group compared with the WTCLP group. Immunohistochemical analysis showed reduced 4-hydroxynonenal, apoptosis, and vascular leakage in the cardiac tissue of Trx-1Tg/+CLP mice compared with mice in the WTCLP group. CONCLUSIONS: Our results indicate that overexpression of Trx-1 attenuates cardiac dysfunction during CLP. The mechanism of action may involve reduction of oxidative stress, apoptosis, and vascular permeability through activation of Trx-1/HO-1 and anti-apoptotic protein survivin.

Overexpression of Thioredoxin1 enhances functional recovery in a mouse model of hind limb ischemia.

BACKGROUND: There is keen interest in finding nonsurgical treatments for peripheral vascular disease (PVD). Previously, we demonstrated that selective activation of Thioredoxin1 (Trx1), a 12-kDa cytosolic protein, initiates redox-dependent signaling and promotes neovascularization after ischemic heart disease. Therefore, Trx1 might possess immense potential to not only treat murine hind limb ischemia (HLI) through effective angiogenesis but also provide PVD patients with nonsurgical therapy to enhance neovascularization and improve blood perfusion. METHODS: To determine whether activation of Trx1 increases blood perfusion in HLI, two different strategies were used-gene therapy and transgenic model system. In adenoviral-mediated gene therapy, 8- to 12-wk-old mice were divided into two groups: (1) control Adeno-LacZ (Ad-LacZ) and (2) Adeno-Thiroedoxin1 (Ad-Trx1). The mice underwent surgical intervention to induce right HLI followed by injection with Ad-LacZ or Ad-Trx1, respectively. In the second strategy, we used wild-type and transgenic mice overexpressing Trx1 (Trx1Tg/+). All the animals underwent Doppler imaging for the assessment of limb perfusion followed by immunohistochemistry and Western blot analysis. RESULTS: Significant increases in perfusion ratio were observed in all the Trx1 overexpressed groups compared with their corresponding controls. Expressions of heme oxygenase-1, vascular endothelial growth factor, and the vascular endothelial growth factor receptors Flk-1 and Flt-1 were increased in Trx1 overexpressed mice compared with their respective controls. Blood perfusion in the ischemic limb gradually improved and significantly recovered in Trx1Tg/+ and Ad-Trx1 groups compared with their corresponding controls. The capillary and arteriolar density in the ischemic zone were found to be higher in Trx1Tg/+ group compared with wild type. CONCLUSIONS: The overall outcomes of our study demonstrate that Trx1 enhances blood perfusion and increases angiogenic protein expression in a rodent HLI model. These results suggest that Trx1 is a potential target for clinical trials and drug therapy for the treatment of PVD.

Trimodal rescue of hind limb ischemia with growth factors, cells, and nanocarriers: fundamentals to clinical trials.

Peripheral artery disease is a severe medical condition commonly characterized by critical or acute limb ischemia. Gradual accumulation of thrombotic plaques in peripheral arteries of the lower limb may lead to intermittent claudication or ischemia in muscle tissue. Ischemic muscle tissue with lesions may become infected, resulting in a non-healing wound. Stable progression of the non-healing wound associated with severe ischemia might lead to functional deterioration of the limb, which, depending on the severity, can result in amputation. Immediate rescue of ischemic muscles through revascularization strategies is considered the gold standard to treat critical limb ischemia. Growth factors offer multiple levels of protection in revascularization of ischemic tissue. In this review, the basic mechanism through which growth factors exert their beneficial properties to rescue the ischemic limb is extensively discussed. Moreover, clinical trials based on growth factor and stem cell therapy to treat critical limb ischemia are considered. The clinical utility of stem cell therapy for the treatment of limb ischemia is explained and recent advances in nanocarrier technology for selective growth factor and stem cell supplementation are summarized.

Increased survivability of ischemic skin flap tissue in Flk-1+/- mice by Pellino-1 intervention.

OBJECTIVE: Reduced skin flap survival due to ischemia is a serious concern during reconstructive cosmetic surgery. The absence of VEGF and its receptors during ischemia may lead to flap failure. We identified Peli1, a 46-kDa protein, as a proangiogenic molecule and is directly regulated by VEGF. Therefore, we hypothesized that Peli1 acts downstream of Flk-1/VEGFR2 and aids in skin flap survival during ischemia. METHODS: Scratch and matrigel assays were performed to observe cell proliferation, migration, and tube formation in vitro. Western blot analysis was carried out to detect the phosphorylation of Akt (p-Akt) and MAPKAPK2 (p-MK2) in HUVECs. The translational potential of Peli1 pretreatment in the rescue of skin flap tissue was studied in vivo using Flk-1+/- mice. Animals underwent dorsal ischemic skin flap surgery, and the tissue was collected on day 12 for analysis. RESULTS: Western blot analysis revealed a direct relationship between Peli1 and VEGF, as demonstrated by loss-of-function and gain-of-function studies. In addition, pretreatment with Ad.Peli1 restored the phosphorylation of Akt and MK2 and improved the migration potential of Flk-1-knockdown cells. Ad.Peli1 pretreatment salvaged the ischemic skin flap of Flk-1+/- mice by increasing blood perfusion and reducing the inflammatory response and the extent of necrosis. CONCLUSION: Our findings reveal that Peli1 is a proangiogenic molecule that acts downstream of VEGF-Flk-1 and restores angiogenesis and enhances skin flap survivability.

Correction: Self-assembly of peptides: influence of substrate, pH and medium on the formation of supramolecular assemblies.

Correction for 'Self-assembly of peptides: influence of substrate, pH and medium on the formation of supramolecular assemblies' by Priyadharshini Kumaraswamy et al., Soft Matter, 2011, 7, 2744-2754.

Engineering a growth factor embedded nanofiber matrix niche to promote vascularization for functional cardiac regeneration.

The major loss of tissue extracellular matrix (ECM) after myocardial ischemia is a serious burden that gradually leads to heart failure. Due to lack of available treatment methods to restore the cardiac function, various research strategies have come up to treat the ischemic myocardium. However these have met with limited success due to the complexity of the cardiac tissue, which exhibits a nanofibrous collagenous matrix with spatio-temporal localization of a combination of growth factors. To mimic the topographical and chemical cues of the natural cardiac tissue, we have fabricated a growth factor embedded nanofibrous scaffold through electrospinning. In our previous work, we have reported a nanofibrous matrix made of PLCL and PEOz with an average diameter of 500 nm. The scaffold properties were specifically characterized in vitro for cardio-compatibility. In the present study, we have loaded dual growth factors VEGF and bFGF in the nanofiber matrix and investigated its suitability for cardiac tissue engineering. The encapsulation and release of dual growth factors from the matrix were studied using XPS and ELISA. Bioactivity of the loaded growth factors towards proliferation and migration of endothelial cells (HUVECs) was evaluated through MTS and Boyden chamber assays respectively. The efficiency of growth factors on the nanofibrous matrix to activate signaling molecules was studied in HUVECs through gene expression analysis. Preclinical evaluation of the growth factor embedded nanofibrous patch in a rabbit acute myocardial infarction (AMI) model was studied and cardiac function assessment was made through ECG and echocardiography. The evidence for angiogenesis in the patch secured regions was analyzed through histopathology and immunohistochemistry. Our results confirm the effectiveness of growth factor embedded nanofiber matrix in restoration of cardiac function after ischemia when compared to conventional patch material thereby exhibiting promise as a valuable therapeutic solution to treat ischemic disorders.

Multidimensional nanofibrous scaffolds of poly(lactide-co-caprolactone) and poly(ethyl oxazoline) with improved features for cardiac tissue engineering.

AIM: The aim of the study is to develop scaffolds that mimic native tissue properties for effective regeneration of the myocardium, which is affected by the gradual thinning of left ventricular tissue after an infarction. MATERIALS & METHODS: Heterogenous nanofibrous scaffolds made of poly(lactide-co-caprolactone) and poly(ethyl oxazoline) were characterized for physico-chemical properties. The biocompatibility of the scaffolds was evaluated by studying the adhesion, proliferation and differentiation of H9c2 cells. RESULTS: The scaffolds mimic the cardiac extracellular matrix and showed enhanced tensile strength, improved cell compatibility along with the expression of cardiac marker proteins. CONCLUSION: Our experimental data confirmed the importance of native tissue architecture and mechanical strength for improved cell response in cardiac tissue engineering.

Polymeric scaffold aided stem cell therapeutics for cardiac muscle repair and regeneration.

The constantly expanding repository of novel polymers and stem cells has opened up new vistas in the field of cardiac tissue engineering. Successful regeneration of the complex cardiac tissue mainly centres on the appropriate scaffold material with topographical features that mimic the native environment. The integration of stem cells on these scaffolds is expected to enhance the regeneration potential. This review elaborates on the interplay of these vital factors in achieving the functional cardiac tissue. The recent advances in polymers, nanocomposites, and stem cells from different sources are highlighted. Special emphasis is laid on the clinical trials involving stem cells and the state-of-the-art materials to obtain a balanced perspective on the translational potential of this strategy.

Living cardiac patch: the elixir for cardiac regeneration.

INTRODUCTION: A thorough understanding of the cellular and muscle fiber orientation in left ventricular cardiac tissue is of paramount importance for the generation of artificial cardiac patches to treat the ischemic myocardium. The major challenge faced during cardiac patch engineering is to choose a perfect combination of three entities; cells, scaffolds and signaling molecules comprising the tissue engineering triad for repair and regeneration. AREAS COVERED: This review provides an overview of various scaffold materials, their mechanical properties and fabrication methods utilized in cardiac patch engineering. Stem cell therapies in clinical trials and the commercially available cardiac patch materials were summarized in an attempt to provide a recent perspective in the treatment of heart failure. Various tissue engineering strategies employed thus far to construct viable thick cardiac patches is schematically illustrated. EXPERT OPINION: Though many strategies have been proposed for fabrication of various cardiac scaffold materials, the stage and severity of the disease condition demands the incorporation of additional cues in a suitable scaffold material. The scaffold may be nanofibrous patch, hydrogel or custom designed films. Integration of stem cells and biomolecular cues along with the scaffold may provide the right microenvironment for the repair of unhealthy left ventricular tissue as well as promote its regeneration.