A multiplex immunoprofiling approach for detecting the co-localization of breast cancer biomarkers using a combination of Alexafluor - Quantum dot conjugates and a panel of chromogenic dyes.

There is a plethora of information embedded in a tissue section that the conventional IHC understands only partially. Predictive biomarkers for precision immuno-oncology heavily dependent on the spatial arrangement of cells and the co-expression patterns in the tissue sections. Here we have explored the versatility of indirect multiplex immunofluorescence (mIF) and indirect multiplex immunohistochemistry (mIHC) for the labeling of breast cancer prognostic markers in routinely processed, formalin-fixed paraffin-embedded (FFPE) tissues at high resolution. The multiplex immunohistochemistry protocol utilized sequential staining for the chromogenic immunolabelling of Estrogen Receptor α (ERα) or Progesterone Receptor (PR), Human Epidermal Growth Factor Receptor 2 (HER2), and Nucleoside diphosphate kinase 1 (NM23) by multicolor chromogens in different combinations. A feasible workflow for multiplex immunofluorescence was also effectively standardized for ERα, PR, and HER2 using combinations of commercially available Alexa Fluor and Quantum dots semiconductor nanocrystal conjugated secondary antibodies. Multiplex chromogenic immunolabeling revealed differential expression of the markers on the same slide. Kappa statistics revealed perfect agreement with uniplex immunohistochemistry. For multiplex fluorescence approach, surface receptor detection using Quantum dots and Alexa fluor dyes for cytoplasmic or nuclear markers performed well for profiling multiple co-localized biomarkers on a single paraffin tissue section. The technique developed reveals additional information such as co-expression, spatial relationships, and tumor heterogeneity, providing a deeper insight into developing combinatorial therapeutic strategies in clinical care. This high throughput workflow complements the outcomes of traditional IHC while saving tissue, time, labour, and reagents.

Co-expression of galectin-3 and vimentin in triple negative breast cancer cells promotes tumor progression, metastasis and survival.

BACKGROUND: Lack of druggable targets and complex expression heterogeneity of known targets is common among TNBC subtypes. An enhanced expression of galectin-3 in TNBCs has already been documented. We have observed a tumor progression-dependent galectin-3 expression in TNBCs compared to adjacent epithelium and non TNBCs. OBJECTIVE: To unravel the association of galectin- 3 in tumor progression, aggressiveness and drug resistance in TNBC patients. METHODS: Galectin-3 expression in 489 breast cancer tissues was correlated with clinicopathological features and the results were validated in cell lines and mouse model by silencing galectin-3 using shRNA and the proteins were profiled by western blot and qRT-PCR. Protein interaction was analyzed by GFP Trap and Mass spectrometry. RESULTS: Galectin-3 expression correlated with tumor stage in TNBC and a lower galectin-3 expression was associated with poor patient survival. The positive correlation between galectin-3, vimentin and CD44 expression, pinpoints galectin-3 contribution to epithelial to mesenchymal transition, drug resistance and stemness. Vimentin was found as an interacting partner of galectin-3. Duplexing of galecin-3 and vimentin in patient samples revealed the presence of tumor cells co-expressing both galectin-3 and vimentin. In vitro studies also showed its role in tumor cell survival and metastatic potential, elementary for tumor progression. In vivo studies further confirmed its metastatic potential. CONCLUSIONS: Tumor progression dependent expression pattern of galectin 3 was found to indicate prognosis. Co-expression of galectin-3 and vimentin in tumor cells promotes tumor dissemination, survival and its metastatic capability in TNBCs.

Multi-Layered PLGA-PEI Nanoparticles Functionalized with TKD Peptide for Targeted Delivery of Pep5 to Breast Tumor Cells and Spheroids.

Purpose: Peptide-based therapy is a promising strategy for cancer treatment because of its low drug resistance. However, the major challenge is their inability to target cancer cells specifically. So, a targeted nano-delivery system that could deliver therapeutic peptides selectively to cancer cells to stimulate their action is highly desirable. This study aims to deliver the antitumor peptide, Pep5, to breast tumor cells selectively using a targeting peptide functionalised multi-layered PLGA-PEI nanoparticles. Methods: In this study, Pep5 entrapped PLGA-PEI (Pep5-PPN) dual layered nanoparticles were developed. These nanoparticles were decorated with TKD (Pep5-TPPN) on their surface for site-specific delivery of Pep5 to breast tumor cells. The particles were then characterized using various instrumental analyses. In vitro cytotoxicity of the particles was evaluated in estrogen receptor positive (ER+ve) and triple negative breast cancer (TNBC) cells. An ex vivo tumor spheroid model was used to analyze the antitumor activity of the particles. Results: Uniformly round Pep5-TPPN particles were synthesized with an average diameter of 420.8 ± 14.72 nm. The conjugation of PEI over Pep5-PLGA nanoparticles shifted the zeta potential from -11.6 ± 2.16 mV to +20.01 ± 2.97 mV. In vitro cytotoxicity analysis proved that TKD conjugation to nanoparticles enhanced the antitumor activity of Pep5 in tested breast cancer cells. Pep5-TPPN induced cytoskeletal damage and apoptosis in the tested cells, which showed that the mechanism of action of Pep5 is conserved but potentiated. Active targeting of Pep5 suppressed the tumor growth in ex vivo spheroid models. Conclusion: A multi-layered nanoparticle functionalized with dual peptide was fabricated for active tumor targeting, which stimulated Pep5 activity to reduce the tumor growth in vitro and ex vivo.

Correction: Calpain and Reactive Oxygen Species Targets Bax for Mitochondrial Permeabilisation and Caspase Activation in Zerumbone Induced Apoptosis.

[This corrects the article DOI: 10.1371/journal.pone.0059350.].

Delivery of Small Molecules by Syndiotactic Peptides for Breast Cancer Therapy.

The utilization of peptide-based drug delivery systems has been suboptimal due to their poor proteolytic susceptibility, poor cell permeability, and limited tumor homing capabilities. Earlier attempts in using d-enantiomers in peptide sequences increased proteolytic stability but have compromised the overall penetration capability. We designed a series of peptides (STRAPs) with a syndiotactic polypeptide backbone that can potentially form a spatial array of cationic groups, an important feature that facilitates cellular uptake. The peptides penetrate cell membranes through a combination of active and passive modes. Furthermore, the cellular uptake of the peptides was unaffected by the presence of or treatment with bovine serum and human plasma. The designed peptides successfully delivered methotrexate, an anticancer drug, to the in vitro and in vivo models of breast cancer, with the best performing peptide STRAP-4-MTX conjugate having an EC50 value of 1.34 µM. Peptide drug delivery in mouse xenograft models showed a greater reduction of primary tumor and metastasis of breast cancer, in comparison to methotrexate of the same dose. The in vivo biodistribution assay of the STRAP-4 peptide suggests that the peptide accumulates at the tumor site after 2 h of treatment, and in the absence of tumors, the peptide gets metabolized and excreted from the system.

Mutational landscape and in silico structure models of SARS-CoV-2 spike receptor binding domain reveal key molecular determinants for virus-host interaction.

BACKGROUND: SARS-CoV-2, the causative agent of COVID-19 pandemic is a RNA virus prone to mutations. Formation of a stable binding interface between the Receptor Binding Domain (RBD) of SARS-CoV-2 Spike (S) protein and Angiotensin-Converting Enzyme 2 (ACE2) of host is pivotal for viral entry. RBD has been shown to mutate frequently during pandemic. Although, a few mutations in RBD exhibit enhanced transmission rates leading to rise of new variants of concern, most RBD mutations show sustained ACE2 binding and virus infectivity. Yet, how all these mutations make the binding interface constantly favourable for virus remain enigmatic. This study aims to delineate molecular rearrangements in the binding interface of SARS-CoV-2 RBD mutants. RESULTS: Here, we have generated a mutational and structural landscape of SARS-CoV-2 RBD in first six months of the pandemic. We analyzed 31,403 SARS-CoV-2 genomes randomly across the globe, and identified 444 non-synonymous mutations in RBD that cause 49 distinct amino acid substitutions in contact and non-contact amino acid residues. Molecular phylogenetic analysis suggested independent emergence of RBD mutants. Structural mapping of these mutations on the SARS-CoV-2 Wuhan reference strain RBD and structural comparison with RBDs from bat-CoV, SARS-CoV, and pangolin-CoV, all bound to human or mouse ACE2, revealed several changes in the interfacial interactions in all three binding clusters. Interestingly, interactions mediated via N487 residue in cluster-I and Y449, G496, T500, G502 residues in cluster-III remained largely unchanged in all RBD mutants. Further analysis showed that these interactions are evolutionarily conserved in sarbecoviruses which use ACE2 for entry. Importantly, despite extensive changes in the interface, RBD-ACE2 stability and binding affinities were maintained in all the analyzed mutants. Taken together, these findings reveal how SARS-CoV-2 uses its RBD residues to constantly remodel the binding interface. CONCLUSION: Our study broadly signifies understanding virus-host binding interfaces and their alterations during pandemic. Our findings propose a possible interface remodelling mechanism used by SARS-CoV-2 to escape deleterious mutations. Future investigations will focus on functional validation of in-silico findings and on investigating interface remodelling mechanisms across sarbecoviruses. Thus, in long run, this study may provide novel clues to therapeutically target RBD-ACE2 interface for pan-sarbecovirus infections.

Molecular hybridization combining tumor homing and penetrating peptide domains for cellular targeting.

A complete peptide-based drug delivery unit has been designed with a tumor homing domain chemically linked to a syndiotactic cell-penetrating domain. The designed peptides were synthesized, characterized, and tested in vitro for cellular uptake and cytotoxicity evaluation. The differential uptake, cellular internalization, negligible hemotoxicity, selective toxicity to MDA-MB-231 breast cancer cells, and the superior penetration in three-dimensional MDA-MB-231 tumorospheres confirm their utility as a promising delivery vector.

Revealing the stability of CuWO4/g-C3N4 nanocomposite for photocatalytic tetracycline degradation from the aqueous environment and DFT analysis.

Graphitic carbon nitride (g-C3N4) is an emerging metal-free photocatalyst, however, engineering the photocatalytic efficiency for the effective degradation of hazardous molecules is still challenging. An unstable and low bandgap CuWO4 was composited with g-C3N4 to achieve synergistic benefits of tuning the visible light responsiveness and stability of CuWO4. CuWO4/g-C3N4 nanocomposite exhibited a relatively high visible light absorption region and the bandgap was modified from 2.77 to 2.53 eV evidenced via UV-DRS. Moreover, the fast electron transfer rate was observed with CuWO4/g-C3N4 nanocomposite as confirmed using PL and photocurrent studies. XRD, FT-IR, and HR-TEM analyses signified the formation of CuWO4/g-C3N4 nanocomposite. CuWO4/g-C3N4 nanocomposite showed enhanced photocatalytic degradation of Tetracycline (TC) about ∼7.4 fold greater than pristine g-C3N4 in 120 min. Notably, the OH• and •O2- radicals played a most significant role in photocatalytic TC degradation. Furthermore, the energy band structure, density of state, and Bader charge analyses of these molecules were performed.

Plasmodium falciparum LipB mutants display altered redox and carbon metabolism in asexual stages and cannot complete sporogony in Anopheles mosquitoes.

Malaria is still one of the most important global infectious diseases. Emergence of drug resistance and a shortage of new efficient antimalarials continue to hamper a malaria eradication agenda. Malaria parasites are highly sensitive to changes in the redox environment. Understanding the mechanisms regulating parasite redox could contribute to the design of new drugs. Malaria parasites have a complex network of redox regulatory systems housed in their cytosol, in their mitochondrion and in their plastid (apicoplast). While the roles of enzymes of the thioredoxin and glutathione pathways in parasite survival have been explored, the antioxidant role of α-lipoic acid (LA) produced in the apicoplast has not been tested. To take a first step in teasing a putative role of LA in redox regulation, we analysed a mutant Plasmodium falciparum (3D7 strain) lacking the apicoplast lipoic acid protein ligase B (lipB) known to be depleted of LA. Our results showed a change in expression of redox regulators in the apicoplast and the cytosol. We further detected a change in parasite central carbon metabolism, with lipB deletion resulting in changes to glycolysis and tricarboxylic acid cycle activity. Further, in another Plasmodium cell line (NF54), deletion of lipB impacted development in the mosquito, preventing the detection of infectious sporozoite stages. While it is not clear at this point if the observed phenotypes are linked, these findings flag LA biosynthesis as an important subject for further study in the context of redox regulation in asexual stages, and point to LipB as a potential target for the development of new transmission drugs.

Transitional dynamics of cancer stem cells in invasion and metastasis.

At the onset, few cancer cells amidst the tumor bulk, identified as cancer stem cells (CSCs) or early disseminated cancer cells (eDCCs) are capable of survival post conventional therapy and persist as minimal residual disease (MRD). Metastatic subclones emerge both early and late in the life of primary tumor ensuing an ongoing regional clonal evolution of progenitor cells in metastatic and primary tumors. In the last decade, multiple studies proposed various identities of stem-like cells that undergo transitions to adapt to the changing microenvironment as the disease progresses. This review advocates with substantial evidence the dynamic model of tumor propagation by exploring the specific cell types, reversible phenotypic plasticity between the tumorigenic leader seeds and the supporting follower cancer cells both in circulation and in solid tissue to accurately decipher tumor promoting clones and its role in metastatic dissemination and tumor re-growth. (142 words).

Enhancement of microwave absorption bandwidth of MXene nanocomposites through macroscopic design.

MXene, the new family of two-dimensional materials having numerous nanoscale layers, is being considered as a novel microwave absorption material. However, MXene/functionalized MXene-loaded polymer nanocomposites exhibit narrow reflection loss (RL) bandwidth (RL less than or equal to -10 dB). In order to enhance the microwave absorption bandwidth of MXene hybrid-matrix materials, for the first time, macroscopic design approach is carried out for TiO2-Ti3C2Tx MXene and Fe3O4@TiO2-Ti3C2Tx MXene hybrids through simulation. The simulated results indicate that use of pyramidal meta structure of MXene can significantly tune the RL bandwidth. For optimized MXene hybrid-matrix materials pyramid pattern, the bandwidth enhances to 3-18 GHz. Experimental RL value well matched with the simulated RL. On the other hand, the optimized Fe3O4@TiO2-Ti3C2Tx hybrid exhibits two specific absorption bandwidths (minimum RL value - -47 dB). Compared with other two-dimensional nanocomposites such as graphene or Fe3O4-graphene, MXene hybrid-matrix materials show better microwave absorption bandwidth in macroscopic pattern.

Polaron and bipolaron induced charge carrier transportation for enhanced photocatalytic H2 production.

Photocatalysis is one of the facile approaches for efficient solar energy conversion and storage. However, rapid charge carrier recombination considerably decreases solar to energy conversion efficiency. Herein, polaron and bipolaron rich polypyrrole (PPy) has been utilized as a solid support for effective photogenerated charge carrier separation. Simple oxidative polymerization using a high concentration of ammonium persulfate (APS) induces radical cation/bipolaron formation in PPy due to the cleavage of π-bonds as confirmed by electron paramagnetic resonance spectroscopy (EPR). The formation of radical cations led to an increase of the dielectric constant which retards the charge carrier recombination and thereby enhances the conductivity. Moreover, the polarons and bipolarons induced charge carrier separation in photocatalytic H2 production was studied with the well-known g-C3N4 photocatalyst. It is worth mentioning that compared to bare g-C3N4, the PPy supported system showed a drastically enhanced photocatalytic H2 production rate. A maximum H2 production rate of 1851 µmoles per g is achieved, which is ∼51 times higher than that of the bare g-C3N4 catalyst due to efficient charge carrier separation assisted by radical cations/bipolarons. Thus, utilizing this simple polaron and bipolaron rich PPy solid support could be an effective strategy and alternative for using noble metal cocatalysts to enhance charge carrier separation.

Inhibition of Resistance-Refractory P. falciparum Kinase PKG Delivers Prophylactic, Blood Stage, and Transmission-Blocking Antiplasmodial Activity.

The search for antimalarial chemotypes with modes of action unrelated to existing drugs has intensified with the recent failure of first-line therapies across Southeast Asia. Here, we show that the trisubstituted imidazole MMV030084 potently inhibits hepatocyte invasion by Plasmodium sporozoites, merozoite egress from asexual blood stage schizonts, and male gamete exflagellation. Metabolomic, phosphoproteomic, and chemoproteomic studies, validated with conditional knockdown parasites, molecular docking, and recombinant kinase assays, identified cGMP-dependent protein kinase (PKG) as the primary target of MMV030084. PKG is known to play essential roles in Plasmodium invasion of and egress from host cells, matching MMV030084's activity profile. Resistance selections and gene editing identified tyrosine kinase-like protein 3 as a low-level resistance mediator for PKG inhibitors, while PKG itself never mutated under pressure. These studies highlight PKG as a resistance-refractory antimalarial target throughout the Plasmodium life cycle and promote MMV030084 as a promising Plasmodium PKG-targeting chemotype.

Pan-active imidazolopiperazine antimalarials target the Plasmodium falciparum intracellular secretory pathway.

A promising new compound class for treating human malaria is the imidazolopiperazines (IZP) class. IZP compounds KAF156 (Ganaplacide) and GNF179 are effective against Plasmodium symptomatic asexual blood-stage infections, and are able to prevent transmission and block infection in animal models. But despite the identification of resistance mechanisms in P. falciparum, the mode of action of IZPs remains unknown. To investigate, we here combine in vitro evolution and genome analysis in Saccharomyces cerevisiae with molecular, metabolomic, and chemogenomic methods in P. falciparum. Our findings reveal that IZP-resistant S. cerevisiae clones carry mutations in genes involved in Endoplasmic Reticulum (ER)-based lipid homeostasis and autophagy. In Plasmodium, IZPs inhibit protein trafficking, block the establishment of new permeation pathways, and cause ER expansion. Our data highlight a mechanism for blocking parasite development that is distinct from those of standard compounds used to treat malaria, and demonstrate the potential of IZPs for studying ER-dependent protein processing.

Molecular Networking and Whole-Genome Analysis Aid Discovery of an Angucycline That Inactivates mTORC1/C2 and Induces Programmed Cell Death.

Rediscovery of known compounds and time consumed in identification, especially high molecular weight compounds with complex structure, have let down interest in drug discovery. In this study, whole-genome analysis of microbe and Global Natural Products Social (GNPS) molecular networking helped in initial understanding of possible compounds produced by the microbe. Genome data revealed 10 biosythethic gene clusters that encode for secondary metabolites with anticancer potential. NMR analysis of the pure compound revealed the presence of a four-ringed benz[a]anthracene, thus confirming angucycline; molecular networking further confirmed production of this class of compounds. The type II polyketide synthase gene identified in the microbial genome was matched with the urdamycin cluster by BLAST analysis. This information led to ease in identification of urdamycin E and a novel natural derivative, urdamycin V, purified from Streptomyces sp. OA293. Urdamycin E (Urd E) induced apoptosis and autophagy in cancer cell lines. Urd E exerted anticancer action through inactivation of the mTOR complex by preventing phosphorylation at Ser 2448 and Ser 2481 of mTORC1 and mTORC2, respectively. Significant reduction in phosphorylation of the major downstream regulators of both mTORC1 (p70s6k and 4e-bp1) and mTORC2 (Akt) were observed, thus further confirming complete inhibition of the mTOR pathway. Urd E presents itself as a novel mTOR inhibitor that employs a novel mechanism in mTOR pathway inhibition.

Peptide-based delivery vectors with pre-defined geometrical locks.

Design of peptide-based targeted delivery vectors with attributes of specificity and selective cellular targeting by fixing their topology and resulting electrostatic fingerprint is the objective of this study. We formulated our peptide design platform by utilizing the possibilities of side-chain induced geometric restrictions in a typical peptide molecule. Conceptually, we locked the conformation of the RGD/NGR motif of tumor homing peptides (THPs) by mutating glycine in these motifs with d-proline and tailed the peptides with a syndiotactic amphipathic segment for cellular penetration. The designed peptides were synthesized, characterized, and tested in vitro on various cell lines, including breast cancer (MDA-MB-231), cervical cancer (HeLa), osteosarcoma (U2-OS) and non-cancer mammary epithelial cells (MCF-10A), by flow cytometry and confocal microscopy. The results showed differential cellular uptake in different cell types, as a result of the distinct electrostatic fingerprint encoded in their design. The uptake of serum pre-treated peptides by cells reveals the retention of peptide activity even after the incubation with serum. In addition, peptide-methotrexate (MTX) conjugates compared to the methotrexate drug showed enhanced apoptotic cell death in MTX-resistant MDA-MB-231 cells, indicating the increase in MTX bioavailability.

Untargeted metabolomics reveals alterations in metabolites of lipid metabolism and immune pathways in the serum of rats after long-term oral administration of Amalaki rasayana.

Amalaki rasayana, a traditional preparation, is widely used by Ayurvedic physicians for the treatment of inflammatory conditions, cardiovascular diseases, and cancer. Metabolic alterations induced by Amalaki rasayana intervention are unknown. We investigated the modulations in serum metabolomic profiles in Wistar rats following long-term oral administration of Amalaki rasayana. Global metabolic profiling was performed of the serum of rats administered with either Amalaki rasayana (AR) or ghee + honey (GH) for 18 months and control animals which were left untreated. Amalaki rasayana components were confirmed from AR extract using HR-LCMS analysis. Significant reductions in prostaglandin J2, 11-dehydrothromboxane B2, and higher levels of reduced glutathione and glycitein metabolites were observed in the serum of AR administered rats compared to the control groups. Eleven different metabolites classified as phospholipids, glycerophospholipids, glucoside derivatives, organic acids, and glycosphingolipid were exclusively observed in the AR administered rats. Pathway analysis suggests that altered metabolites in AR administered rats are those associated with different biochemical pathways of arachidonic acid metabolism, fatty acid metabolism, leukotriene metabolism, G-protein mediated events, phospholipid metabolism, and the immune system. Targeted metabolomics confirmed the presence of gallic acid, ellagic acid, and arachidonic acid components in the AR extract. The known activities of these components can be correlated with the altered metabolic profile following long-term AR administration. AR also activates IGF1R-Akt-Foxo3 signaling axis in heart tissues of rats administered with AR. Our study identifies AR components that induce alterations in lipid metabolism and immune pathways in animals which consume AR for an extended period.

Chronic Pressure Overload Results in Deficiency of Mitochondrial Membrane Transporter ABCB7 Which Contributes to Iron Overload, Mitochondrial Dysfunction, Metabolic Shift and Worsens Cardiac Function.

We examined the hitherto unexplored role of mitochondrial transporters and iron metabolism in advancing metabolic and mitochondrial dysfunction in the heart during long term pressure overload. We also investigated the link between mitochondrial dysfunction and fluctuation in mitochondrial transporters associated with pressure overload cardiac hypertrophy. Left ventricular hypertrophy (LVH) was induced in 3-month-old male Wistar rats by constriction of the aorta using titanium clips. After sacrifice at the end of 6 and 15 months after constriction, tissues from the left ventricle (LV) from all animals were collected for histology, biochemical studies, proteomic and metabolic profiling, and gene and protein expression studies. LV tissues from rats with LVH had a significant decrease in the expression of ABCB7 and mitochondrial oxidative phosphorylation (mt-OXPHOS) enzymes, an increased level of lipid metabolites, decrease in the level of intermediate metabolites of pentose phosphate pathway and elevated levels of cytoplasmic and mitochondrial iron, reactive oxygen species (ROS) and autophagy-related proteins. Knockdown of ABCB7 in H9C2 cells and stimulation with angiotensin II resulted in increased ROS levels, ferritin, and transferrin receptor expression and iron overload in both mitochondria and cytoplasm. A decrease in mRNA and protein levels of mt-OXPHOS specific enzymes, mt-dynamics and autophagy clearance and activation of IGF-1 signaling were also seen in these cells. ABCB7 overexpression rescued all these changes. ABCB7 was found to interact with mitochondrial complexes IV and V. We conclude that in chronic pressure overload, ABCB7 deficiency results in iron overload and mitochondrial dysfunction, contributing to heart failure.

Engineered Human Adipose-Derived Stem Cells Inducing Endothelial Lineage and Angiogenic Response.

IMPACT STATEMENT: With respect to the persistent hunt for a cytocompatible, translational, reproducible, and effective approach in engineering primary human adipose-derived mesenchymal stromal cells (hADMSCs), we demonstrate the application of Neon® Transfection System in adequate transient delivery of angiogenic factors. The study presents functional assessment of this approach in vitro, with two notable outcomes at translational perspective; (1) Bioengineered hADMSCs secretome does induce endothelial lineage commitment of stem cells at both transcriptional and translational levels and (2) Combinatorial delivery of vascular endothelial growth factor A and hypoxia-inducible factor-1α by bioengineered hADMSCs enhance upregulation of endothelial cell proliferation, migration-associated wound closure, and endothelial tube formation with augmented Flk-1 expression, as compared with their independent actions. The methods described in this study paves way for in vivo evaluation on identification of appropriate chronic wound models and subsequently for clinical translation. The technology developed also has application in vascularization of tissue-engineered constructs.