Efficient wound healing remains a formidable medical challenge in clinical practice, due to the prevalence of skin defects arising from diverse etiological factors. It is indisputable that epidermal growth factor (EGF) plays a pivotal role in wound repair. However, its clinical application through recombinant proteins encounters challenges, including a short half-life in vivo and high production costs. Addressing these limitations, recent advancements in chemically modified mRNA (cmRNA) technologies offer a promising alternative. This study explores the utilisation of cmRNA in a biocompatible citrate-saline formulation to encode EGF for therapeutic purposes, capitalising on the advantages of cmRNA's inherent stability and the formulation's compatibility with biological systems. CmRNA demonstrated high transfection efficiency in human immortalised keratinocyte (HaCaT) and normal human dermal fibroblasts (NHDF) cells (93.97% ± 1.25% and 90.37% ± 0.97%, respectively), resulting in efficient production of biologically active EGF protein. In vitro, EGF cmRNA significantly promoted HaCaT and NHDF cell cycle, proliferation and migration. In vivo, in vivo imaging system (IVIS) imaging of murine skin confirmed localised and sustained expression of Luciferase cmRNA, with signals detectable up to 11 days post-injection. Immunohistochemistry revealed protein expression in both epidermal and dermal layers as early as 1 h post-injection, peaking at 48 h, further corroborated by enzyme-linked immunosorbent assay (ELISA). In a full-thickness skin defect mouse model, EGF cmRNA significantly accelerated wound healing, with superior re-epithelialisation observed compared to controls by Day 6. Mitogen-activated protein kinase (MEK)/Extracellular signal-regulated kinase (ERK) and Ki67 mRNA expression levels were markedly increased, both in vitro and in vivo. By Day 14, histological and immunohistochemical analyses revealed that EGF cmRNA outperformed recombinant human EGF (rhEGF), as indicated by enhanced formation of hair follicles and cutaneous glands, better-organised collagen fibres, and a reduced collagen Type I/III ratio. No adverse effects were observed in major organs, confirming cmRNA's biosafety. These results highlight the therapeutic potential of EGF-encoding cmRNA as an effective and safe alternative for enhancing wound healing.

An intricate biochemical system of coordinated cellular reactions is involved in restoring damaged tissue after wounds. In chronic wounds, such as diabetic foot ulcers, poor angiogenesis is a common stumbling block due to elevated glucose levels, increased proteolytic enzyme activity, and decreased production of growth factors. While various strategies, including modulation of inflammatory cells, administration of growth factors, and therapies involving stem cells or genes, have been explored to promote angiogenesis, they often suffer from limitations such as poor biodistribution, immunological rejection, administration/dosing, and proteolytic instability. Glycosaminoglycans, such as heparan sulfate, facilitate growth factor interactions with their receptors to induce angiogenic signaling, but their exogenous administration is hindered by poor stability, low serum half-life, and immunogenicity. Cyclic peptides, known for their structural stability and specificity, offer a promising alternative for inducing angiogenesis upon functional modifications. In this work, we developed heparan sulfate (HS)-mimetic cyclic peptide nanotubes (CPNTs) grafted with bioactive groups to enhance angiogenesis without using exogenous growth factors, drugs, or supplements. These CPNTs incorporate glutamic acid, serine, and sulfonated lysine to mimic the functional groups in heparin. The sulfonated cyclic hexapeptide nanotubes developed from DPro-LTrp-DLeu-LSer-DGlu-LLys demonstrated significant proangiogenic activity in HUVECs under hyperglycemic conditions; enhanced endothelial cell motility, invasion, and tube formation; and upregulation of proangiogenic genes and proteins. These HS-mimicking nanotubes have shown a strong potential for promoting impaired angiogenesis, without incorporating exogenous growth factors, and show strong potential in treating diabetic wounds. To the best of our knowledge, this is the first report on the use of HS-mimetic proangiogenic cyclic peptide nanotubes for diabetic wound healing.

Diabetic wounds are complicated by underlying peripheral vasculopathy. Reliance on vascular endothelial growth factor (VEGF) therapy to improve perfusion makes logical sense, yet clinical study outcomes on rescuing diabetic wound vascularization have yielded disappointing results. Our previous work has identified that low endothelial phospholipase Cγ2 (PLCγ2) expression hinders the therapeutic effect of VEGF on the diabetic ischemic limb. In this work, guided by single-cell RNA sequencing of human wound edge, we test the efficacy of gene-targeted therapeutic demethylation intending to improve VEGF-mediated neovascularization. PLCγ2 expression was diminished in all five identified diabetic wound-edge endothelial subclusters encompassing arterial, venous, and capillary cells. Such low expression was associated with hypermethylated PLCγ2 promoter. PLCγ2 promoter was also hypermethylated at murine diabetic ischemic wound edge. To specifically demethylate endothelial PLCγ2 promoter during VEGF therapy, a CRISPR-dCas9-based demethylation cocktail was delivered to the ischemic wound edge using tissue nanotransfection (TNT) technology. Demethylation-based upregulation of PLCγ2 during VEGF therapy improved wound tissue blood flow with an increased abundance of von Willebrand factor (vWF)+/PLCγ2+ vascular tissue elements by activating p44/p42-mitogen-activated protein kinase (MAPK) → hypoxia-inducible factor [HIF]-1α pathway. Taken together, TNT-based delivery of plasmids to demethylate the PLCγ2 gene promoter activity led to significant improvements in VEGF therapy for cutaneous diabetic wounds, resulting in better perfusion and accelerated wound closure.

Obesity and associated metabolic disturbances worsen brain ischemia outcome. High fat diet (HFD)-fed mice are obese and have cerebrovascular remodeling and worsened brain ischemia outcome. We determined whether HFD-induced cerebrovascular remodeling impaired reperfusion to the ischemic penumbra. Six-week-old C57BL/6J or matrix metalloprotease-9 knockout (MMP-9-/-) mice were on HFD or regular diet (RD) for 12 to 14 months before a 60-min left middle cerebral arterial occlusion (MCAO). Photoacoustic microscopy was performed at left cerebral frontal cortex. HFD increased cerebrovascular density and tortuosity in C57BL/6J mice but not in MMP-9-/- mice. Blood flow to the ischemic penumbra slowly recovered but did not reach the baseline 2 h after MCAO in RD-fed mice. Oxygen extraction fraction was increased to maintain cerebral metabolic rate of oxygen (CMRO2) throughout brain ischemia and reperfusion period. This blood flow recovery was worsened in HFD-fed mice, leading to decreased CMRO2. MMP-9-/- attenuated these HFD effects. HFD increased MMP-9 activity and interleukin 1β. Pyrrolidine dithiocarbamate, an anti-inflammatory agent, abolished the HFD effects. Interleukin 1β increased MMP-9 activity. In summary, HFD induces cerebrovascular remodeling, leading to worsened recovery of blood supply to the ischemic penumbra to contribute to poor outcome after brain ischemia. Neuroinflammation may activate MMP-9 in HFD-fed mice.

Gene therapy, which involves the delivery of genetic material into cells to correct an underlying genetic problem, has emerged as a promising approach for treating various conditions. To promote research in this rapidly evolving field, we developed the Gene Therapy Omnibus (GTO) (http://www.inbirg.com/gto/), a comprehensive resource containing detailed clinical trial data and molecular information related to gene therapy. The GTO includes 6333 clinical trial records and 3466 transcriptome profiles, with information on 614 altered genes and 22 types of gene therapy, including DNA therapies, RNA therapies and genetically-modified cell therapies. For each gene therapy product in a clinical trial, detailed information, such as altered gene name, structural components, indication, vector information, phase of the clinical trial, clinical outcomes and adverse effects, is provided when available. Additionally, 345 comparison datasets, including 29 single-cell RNA-sequencing datasets comprising information on both gene therapy and control samples, were established. Differential gene expression and downstream functional enrichment analyses were performed through standardized pipelines to elucidate the molecular alterations induced by gene therapy. The user-friendly interface of the GTO supports efficient data retrieval, visualization and analysis, making it an invaluable resource for researchers and clinicians performing clinical research on gene therapy and the underlying mechanisms.

Proper oxygen delivery through the microvasculature to injury site is essential to ensure the metabolic cascade during wound healing. Adaptation of vascular structure and oxygenation is key to unravel the regulation of blood perfusion, oxygen distribution and new tissue formation. Yet, visualizing micrometabolic responses at large scale in unperturbed living tissue remains challenging. We studied full-thickness excisional wounds in the mouse dorsal skin in vivo using ultrasound-aided spectroscopic large-scale optoacoustic microscopy. Skin layer-specific vascularization is visualized at capillary resolution over centimeter-scale field-of-view in a non-invasive, label-free manner. Different vascular parameters, including oxygenation, diameter and its irregularity, tortuosity and angular alignment, show distinct spatial and temporal variations. Elevated oxygenation is manifested close to the wound at day 4 with the trend accompanied by reduction in diameter over time. Angular alignment increases over time, indicating a more directed blood supply towards the wound. Our observations indicate that wound angiogenesis initiates as capillary sprouting with enlarged newborn vessels and elevated oxygenation around the wound, with the vessels normalizing in size and oxygenation during remodeling. Our study provides insight into micrometabolic profiles surrounding the healing wound, setting the stage for preclinical studies on oxygen delivery mechanisms in pathological skin conditions and during pharmacological interventions.

Preclinical models of cutaneous wound healing can be useful for improving clinical wound care. Oxygen Enhanced Photoacoustic imaging (OE PAI) can measure oxygenation, and Dynamic Contrast Enhanced (DCE) PAI can measure vascular perfusion. We investigated how a combined OE-DCE PAI protocol can measure vascular oxygenation and perfusion to a cutaneous healing model.

We developed a cutaneous "punch" wound model and photographed the wounds to track healing for 9 days. We performed OE-DCE PAI on Day 0, 3, 6, and 9. OE PAI was performed with 21% O2 and 100% O2 breathing gases to measure oxyhemoglobin (HbO2), deoxyhemoglobin (Hb), total hemoglobin (HbT), and oxygen saturation (%sO2), along with changes in these parameters caused by a change in breathing gas (ΔHb, ΔHbO2, ΔHbT, ΔsO2). To perform DCE PAI, indocyanine green (ICG) was administered intravenously while monitoring the PAI signal for 10 min as the agent washed through the wound area, which was used to evaluate the wash-out rate.

The average wound size was significantly smaller only by Day 6. For comparison, OE PAI measured a significant increase in HbO2, Hb, HbT, and %sO2 immediately after creating the wound, which significantly decreased by Day 3 and continued to decrease towards values for normal tissue by Day 9. The vascular wash-out rate significantly increased by Day 3, and continued to increase during the healing process. Notably, the wash-out rate can be assessed at a single PAI absorbance wavelength and by simply comparing signal amplitudes without advanced analysis, which may facilitate clinical translation.

OE-DCE PAI can monitor significant changes in vascular perfusion and oxygenation prior to significant changes in cutaneous wound size. These results establish OE-DCE PAI as a tool for future pre-clinical wound healing studies and eventual clinical translation.

In the last decade, messenger ribonucleic acid (mRNA)-based drugs have gained great interest in both immunotherapy and non-immunogenic applications. This surge in interest can be largely attributed to the demonstration of distinct advantages offered by various mRNA molecules, alongside the rapid advancements in nucleic acid delivery systems. It is noteworthy that the immunogenicity of mRNA drugs presents a double-edged sword. In the context of immunotherapy, extra supplementation of adjuvant is generally required for induction of robust immune responses. Conversely, in non-immunotherapeutic scenarios, immune activation is unwanted considering the host tolerability and high expression demand for mRNA-encoded functional proteins. Herein, mainly focused on the linear non-replicating mRNA, we overview the preclinical and clinical progress and prospects of mRNA medicines encompassing vaccines and other therapeutics. We also highlight the importance of focusing on the host-specific variations, including age, gender, pathological condition, and concurrent medication of individual patient, for maximized efficacy and safety upon mRNA administration. Furthermore, we deliberate on the potential challenges that mRNA drugs may encounter in the realm of disease treatment, the current endeavors of improvement, as well as the application prospects for future advancements. Overall, this review aims to present a comprehensive understanding of mRNA-based therapies while illuminating the prospective development and clinical application of mRNA drugs.

Intervertebral disc degeneration (IVDD) is the primary cause of low back pain, with oxidative stress being a recognized factor that causes its development. Presently, low back pain imposes a significant global economic burden. However, the effectiveness of treatments for IVDD remains extremely limited. Therefore, this study aims to explore innovative and effective IVDD treatments by focusing on oxidative stress as a starting point. In this study, an injectable reactive oxygen species-responsive hydrogel (PVA-tsPBA@SLC7A11 modRNA) is developed, designed to achieve rapid loading and selective release of chemically synthesized modified mRNA (modRNA). SLC7A11 modRNA is specifically used to upregulate the expression of the ferroptosis marker SLC7A11. The local injection of PVA-tsPBA@SLC7A11 modRNA into the degenerated intervertebral disc (IVD) results in the cleavage of PVA-tsPBA, leading to the release of enclosed SLC7A11 modRNA. The extent of SLC7A11 modRNA release is directly proportional to the severity of IVDD, ultimately ameliorating IVDD by inhibiting ferroptosis in nucleus pulposus cells (NPCs). This study proposes an innovative system of PVA-tsPBA hydrogel-encapsulated modRNA, representing a potential novel treatment strategy for patients with early-stage IVDD.

Background: Adipocytes play a crucial role in tissue regeneration, contributing to the restoration of damaged areas and modulating the inflammatory milieu. The modulation of gene expression through chemically modified PPARγ mRNA (PPARγ-modRNA) introduces a sophisticated approach to precisely control adipogenic processes. This study aims to explore the adipogenic potential of the PPARγ-modRNA in 3T3-L1 preadipocytes and its role in wound healing. Materials and Methods: We transfected 3T3-L1 preadipocytes with PPARγ-modRNA to investigate adipogenic differentiation and cellular proliferation in vitro. In vivo, we employed a murine full-thickness skin defect model and compared the effects of modRNA-mediated PPARγ overexpression with control groups. Additionally, we conducted RNA sequencing on luciferase-modified mRNA (LUC) and PPARγ-modRNA-transfected cells (PPAR) for a comprehensive understanding of molecular mechanisms. Results: PPARγ-modRNA significantly enhanced adipogenesis and proliferation in 3T3-L1 preadipocytes in vitro. The injection of PPARγ-modified mRNA led to accelerated wound healing compared to the control groups in vivo. RNA sequencing revealed upregulation of adipogenesis-related genes in the PPAR group, notably associated with the TNF signaling pathway. Subsequently, the KEGG analysis indicated that modRNA-mediated PPARγ overexpression effectively promoted adipogenesis while inhibiting TNF-α-mediated inflammation and cellular apoptosis. Conclusions: This study demonstrates the innovative use of PPARγ-modRNA to induce adipogenesis and expedite wound healing. The nuclear expression of PPARγ through modRNA technology signifies a notable advancement, with implications for future therapeutic strategies targeting adipogenic processes and the inhibition of inflammation in the context of wound healing.

Diabetic foot ulcer (DFU), which is characterised by damage to minute blood vessels or capillaries around wounds, is one of the most serious and dreaded complications of diabetes. It is challenging to repair chronic non-healing DFU wounds. Vascular endothelial growth factor (VEGF) plays an important role in angiogenesis and promotes wound healing in DFU. However, it is difficult to sustainably deliver VEGF to the wound site owing to its poor stability and easy degradation. To overcome this challenge, lipid nanoparticles (LNP) encapsulating circular RNA (circRNA) encoding VEGF-A have been developed to continuously generate and release VEGF-A and accelerate diabetic wound healing. First, VEGF-A circRNA was synthesized using group I intron autocatalysis strategy and confirmed by enzyme digestion, polymerase chain reaction, and sequencing assay. VEGF-A circRNA was encapsulated in ionizable lipid U-105-derived LNP (U-LNP) using microfluidic technology to fabricate U-LNP/VEGF-A circRNA. For comparison, a commercially ionizable lipid ALC-0315-derived LNP (A-LNP) encapsulating circRNA (A-LNP/circRNA) was used. Dynamic light scattering and transmission electron microscopy characterization indicated that U-LNP/circRNA had spherical structure with an average diameter of 108.5 nm, a polydispersity index of 0.22, and a zeta potential of -3.31 mV. The messenger RNA (mRNA) encapsulation efficiency (EE%) of U-LNP was 87.12%. In vitro transfection data confirmed better stability and long-term VEGF-A expression of circRNA compared with linear mRNA. Assessment of cytotoxicity and innate immunity further revealed that U-LNP/circRNA was biocompatible and induced a weak congenital immune response. Cell scratch and angiogenesis tests demonstrated the bioactivity of U-LNP/VEGF-A circRNA owing to its VEGF-A expression. In situ bioluminescence imaging of firefly luciferase (F-Luc) probe and ELISA demonstrated that circRNA had long-term and strong expression of VEGF-A in the first week, and a gradual decrease in the next week at the wound site and surrounding areas. Finally, a diabetic mouse model was used to validate the healing effect of U-LNP/VEGF-A circRNA formulation. The results showed that a single dose of U-LNP/VEGF-A circRNA administered by dripping resulted in almost complete wound recovery on day 12, which was significantly superior to that of U-LNP/VEGF-A linear mRNA, and it also outperformed recombinant human vascular endothelial growth factor (rhVEGF) injection and A-LNP/circRNA dripping. Histological analysis confirmed the healing efficiency and low toxicity of U-LNP/VEGF-A circRNA formulation. Together, VEGF-A circRNA delivered by U-105-derived LNP showed good performance in wound healing, which was ascribed to the long-term expression and continuous release of VEGF-A, and has potential applications for the treatment of diabetic foot ulcer wounds.

Diabetic wounds (DWs) are open sores that can occur anywhere on a diabetic patient's body. They are often complicated by infections, hypoxia, oxidative stress, hyperglycemia, and reduced growth factors and nucleic acids. The healing process involves four phases: homeostasis, inflammation, proliferation, and remodeling, regulated by various cellular and molecular events. Numerous genes and signaling pathways such as VEGF, TGF-β, NF-κB, PPAR-γ, MMPs, IGF, FGF, PDGF, EGF, NOX, TLR, JAK-STAT, PI3K-Akt, MAPK, ERK, JNK, p38, Wnt/β-catenin, Hedgehog, Notch, Hippo, FAK, Integrin, and Src pathways are involved in these events. These pathways and genes are often dysregulated in DWs leading to impaired healing. The present review sheds light on the pathogenesis, healing process, signaling pathways, and genes involved in DW. Further, various therapeutic strategies that target these pathways and genes via nanotechnology are also discussed. Additionally, clinical trials on DW related to gene therapy are also covered in the present review.

Poly(ethylene glycol) (PEG)-lipids are used in Food-and-Drug-Administration-approved lipid nanoparticle (LNP)-RNA drugs, which are safe and effective. However, it is reported that PEG-lipids may also contribute to accelerated blood clearance and rare cases of hypersensitivity; this highlights the utility of exploring PEG-lipid alternatives. Here, it is shown that LNPs containing poly(2-ethyl-2-oxazoline) (PEOZ)-lipids can deliver messenger RNA (mRNA) to multiple cell types in mice inside and outside the liver. In addition, it is reported that LNPs formulated with PEOZ-lipids show reduced clearance from the bloodstream and lower levels of antistealth lipid immunoglobulin Ms than LNPs formulated with PEG-lipids. These data justify further exploration of PEOZ-lipids as alternatives to PEG-lipids in LNP-RNA formulations.

Despite several promising preclinical studies performed over the past two decades, there remains a paucity of market-approved drugs to treat chronic lower extremity wounds in humans. This translational gap challenges our understanding of human chronic lower extremity wounds and the design of wound treatments. Current targeted drug treatments and delivery systems for lower extremity wounds rely heavily on preclinical animal models meant to mimic human chronic wounds. However, there are several key differences between animal preclinical wound models and the human chronic wound microenvironment, which can impact the design of targeted drug treatments and delivery systems. To explore these differences, this review delves into recent new drug technologies and delivery systems designed to address the chronic wound microenvironment. It also highlights preclinical models used to test drug treatments specific for the wound microenvironments of lower extremity diabetic, venous, ischemic, and burn wounds. We further discuss key differences between preclinical wound models and human chronic wounds that may impact successful translational drug treatment design.

Chronic wounds pose a significant global public health challenge due to their suboptimal treatment efficacy caused by bacterial infections and microcirculatory disturbances. Inspired by the biofunctionality of natural skin, an artificial skin (HV@BC@TBG) is bioengineered with bacterial cellulose (BC) sandwiched between photosensitizers (PS) and functionalized living cells. Glucose-modified PS (TBG) and vascular endothelial growth factor (VEGF)-functionalized living cells (HV) are successively modified on each side of BC through biological metabolism and bio-orthogonal reaction. As the outermost layer, the TBG layer can generate reactive oxygen species (ROS) upon light illumination to efficiently combat bacterial infections. The HV layer is the inner layer near the diabetic wound, which servs as a living factory to continuously secrete VEGF to accelerate wound repair by promoting fibroblast proliferation and angiogenesis. The sandwiched structural artificial skin HV@BC@TBG is nontoxic, biocompatible, and demonstrated its ability to significantly accelerate the healing process of infected diabetic wounds, rendering it a promising next-generation medical therapy for chronic wound management.

Exosomes are nano-sized membrane extracellular vesicles which can be released from various types of cells. Exosomes originating from inflammatory or injured cells can have detrimental effects on recipient cells, while exosomes derived from stem cells not only facilitate the repair and regeneration of damaged tissues but also inhibit inflammation and provide protective effects against various diseases, suggesting they may serve as an alternative strategy of stem cells transplantation. Exosomes have a fundamental role in communication between cells, through the transfer of proteins, bioactive lipids and nucleic acids (like miRNAs and mRNAs) between cells. This transfer significantly impacts both the physiological and pathological functions of recipient cells. Nuclear factor erythroid 2-related factor 2 (Nrf2), a transcription factor, is able to mitigate damage caused by oxidative stress and inflammation through various signaling pathways. The positive effects resulting from the activation of the Nrf2 signaling pathway in different disorders have been documented in various types of literature. Studies have confirmed that exosomes derived from stem cells could act as Nrf2 effective agonists. However, limited studies have explored the Nrf2 role in the therapeutic effects of stem cell-derived exosomes. This review provides a comprehensive overview of the existing knowledge concerning the role of Nrf2 signaling pathways in the impact exerted by stem cell exosomes in some common diseases.

Wound healing is a multiphase process with a complex repair mechanism; trauma-repairing products with safety and high efficiency have a great market demand. Egg white peptides (EWP) have various physiological regulatory functions and have been proven efficient in ameliorating skin damage. However, their underlying regulation mechanism has not been revealed. This study further evaluated the EWP ameliorating mechanism by conducting a full-thickness skin wound model. Results demonstrated that EWP administration significantly inhibited the expression of pro-inflammatory and shortened the inflammatory phase. Besides, EWP can accelerate the secretion of growth factors (PDGF, VEGF, and TGF-β1) in skin tissue, significantly increasing the regeneration of granulation tissue and endothelium in the proliferation phase, thereby promoting wound healing. After 400 mg/kg EWP interventions for 13 days postoperation, the wound healing rate reached 90%. The combination of transcriptomic and proteomic analyses demonstrated the ameliorating efficiency effects of EWP on wound healing. EWP mainly participates in the functional network with the PI3K-AKT signaling pathway as the core to accelerate wound healing. These findings suggest a promising EWP-based strategy for accelerating wound healing.

Diabetic foot ulcers (DFU) pose a significant health risk in diabetic patients, with insufficient revascularization during wound healing being the primary cause. This study aimed to assess microvessel sprouting and wound healing capabilities using vascular endothelial growth factor (VEGF-A) and a modified fibroblast growth factor (FGF1).

An ex vivo aortic ring rodent model and an in vivo wound healing model in diabetic mice were employed to evaluate the microvessel sprouting and wound healing capabilities of VEGF-A and a modified FGF1 both as monotherapies and in combination.

The combination of VEGF-A and FGF1 demonstrated increased vascular sprouting in the ex vivo mouse aortic ring model, and topical administration of a combination of VEGF-A and FGF1 mRNAs formulated in lipid nanoparticles (LNPs) in mouse skin wounds promoted faster wound closure and increased neovascularization seven days post-surgical wound creation. RNA-sequencing analysis of skin samples at day three post-wound creation revealed a strong transcriptional response of the wound healing process, with the combined treatment showing significant enrichment of genes linked to skin growth.

f-LNPs encapsulating VEGF-A and FGF1 mRNAs present a promising approach to improving the scarring process in DFU.

Impaired wound healing in diabetes mellitus (DM) is a major health burden on patients, their families, and society. The present study aimed to systematically profile the m6A modification landscape in cutaneous wounds in a diabetic mouse model.

Diabetes was induced in mice through a single intraperitoneal injection of streptozotocin (STZ); a single intraperitoneal injection of PBS was made in control mice for comparisons. Both groups then received an 8-mm diameter, full-thickness dorsal body wound with a biopsy punch. Five days after wound surgery, western blot analysis of harvested wound tissues from both groups was used to assess the expression of m6A-related enzymes. Genome-wide profiling of m6A-tagged transcripts was performed through MeRIP-seq and RNA-seq.

ALKBH5, an m6A eraser, was significantly upregulated, while METTL3, METTL14, and WTAP, m6A writers, were markedly downregulated in the diabetic wounds. Additionally, a total of 1335 m6A peaks were differentially expressed in MeRIP-seq and RNA-seq analyses, with 558 upregulated and 777 downregulated peaks. Finally, there was hypomethylated and hypermethylated differentiation at the gene and transcript levels.

The present study was the first to reveal the m6A landscape in diabetic wounds in an animal model.

This study, by deeply analyzing the role of m6A modifications in diabetic wound healing, provides new insights and understanding into the molecular mechanisms of diabetic wound healing. Future research could further explore how m6A modifications regulate the wound healing process, thereby offering new potential targets for the treatment of diabetic wounds.

Over 100 monoclonal antibodies have been approved by the U.S. Food and Drug Administration (FDA) for clinical use; however, a paucity of knowledge exists regarding the injection site behavior of these formulated therapeutics, particularly the effect of antibody, formulation, and tissue at the injection site. A deeper understanding of antibody behavior at the injection site, especially on blood oxygenation through imaging, will help design improved versions of the therapeutics for a wide range of diseases.

The aim of this research is to understand the dynamics of monoclonal antibodies at the injection site as well as how the antibody itself affects the functional characteristics of the injection site [e.g., blood oxygen saturation (sO 2 )].

We employed triple-wavelength equipped functional photoacoustic imaging to study the dynamics of dye-labeled and unlabeled monoclonal antibodies at the site of injection in a mouse ear. We injected a near-infrared dye-labeled (and unlabeled) human IgG4 isotype control antibody into the subcutaneous space in mouse ears to analyze the injection site dynamics and quantify molecular movement, as well as its effect on local hemodynamics.

We performed pharmacokinetic studies of the antibody in different regions of the mouse body to show that dye labeling does not alter the pharmacokinetic characteristics of the antibody and that mouse ear is a viable model for these initial studies. We explored the movement of the antibody in the interstitial space to show that the bolus area grows by ∼ 300 % over 24 h. We discovered that injection of the antibody transiently reduces the local sO 2 levels in mice after prolonged anesthesia without affecting the total hemoglobin content and oxygen extraction fraction.

This finding on local oxygen saturation opens a new avenue of study on the functional effects of monoclonal antibody injections. We also show the suitability of the mouse ear model to study antibody dynamics through high-resolution imaging techniques. We quantified the movement of antibodies at the injection site caused by the interstitial fluid, which could be helpful for designing antibodies with tailored absorption speeds in the future.

mRNA vaccines have emerged as highly effective strategies in the prophylaxis and treatment of diseases, thanks largely although not totally to their extraordinary performance in recent years against the worldwide plague COVID-19. The huge superiority of mRNA vaccines regarding their efficacy, safety, and large-scale manufacture encourages pharmaceutical industries and biotechnology companies to expand their application to a diverse array of diseases, despite the nonnegligible problems in design, fabrication, and mode of administration. This review delves into the technical underpinnings of mRNA vaccines, covering mRNA design, synthesis, delivery, and adjuvant technologies. Moreover, this review presents a systematic retrospective analysis in a logical and well-organized manner, shedding light on representative mRNA vaccines employed in various diseases. The scope extends across infectious diseases, cancers, immunological diseases, tissue damages, and rare diseases, showcasing the versatility and potential of mRNA vaccines in diverse therapeutic areas. Furthermore, this review engages in a prospective discussion regarding the current challenge and potential direction for the advancement and utilization of mRNA vaccines. Overall, this comprehensive review serves as a valuable resource for researchers, clinicians, and industry professionals, providing a comprehensive understanding of the technical aspects, historical context, and future prospects of mRNA vaccines in the fight against various diseases.

Myocardial infarction, caused by a thrombus or coronary vascular occlusion, leads to irreversible ischaemic injury. Advances in early reperfusion strategies have significantly reduced short-term mortality after myocardial infarction. However, survivors have an increased risk of developing heart failure, which confers a high risk of death at 1 year. The capacity of the injured neonatal mammalian heart to regenerate has stimulated extensive research into whether recapitulation of developmental regeneration programmes may be beneficial in adult cardiovascular disease. Restoration of functional blood and lymphatic vascular networks in the infarct and border regions via neovascularisation and lymphangiogenesis, respectively, is a key requirement to facilitate myocardial regeneration. An improved understanding of the endogenous mechanisms regulating coronary vascular and lymphatic expansion and function in development and in adult patients after myocardial infarction may inform future therapeutic strategies and improve translation from pre-clinical studies. In this review, we explore the underpinning research and key findings in the field of cardiovascular regeneration, with a focus on neovascularisation and lymphangiogenesis, and discuss the outcomes of therapeutic strategies employed to date. © 2023 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Potency is one of the critical quality attributes of biological medicinal products, defining their biological activity. Potency testing is expected to reflect the Mechanism of Action (MoA) of the medicinal product and ideally the results should correlate with the clinical response. Multiple assay formats may be used, both in vitro assays and in vivo models, however, for timely release of the products for clinical studies or for commercial use, quantitative, validated in vitro assays are necessary. Robust potency assays are fundamental also for comparability studies, process validation and for stability testing. Cell and Gene Therapy Products (CGTs, also called Advanced Therapy Medicinal Products, ATMPs) are part of biological medicines, having nucleic acids, viral vectors, viable cells and tissues as starting material. For such complex products potency testing is often challenging and may require a combination of methods to address multiple functional mechanisms of the product. For cells, viability and cell phenotype are important attributes but alone will not be sufficient to address potency. Furthermore, if the cells are transduced with a viral vector, potency probably is related to the expression of the transgene but will also be dependent on the target cells and transduction efficiency/copy number of the transgene in the cells. Genome Editing (GE) together with other cell manipulations can result into multiple changes in the characteristics and activity of the cells, which should be all somehow captured by the potency testing. Non-clinical studies/models may provide valuable support for potency testing, especially for comparability testing. However, sometimes lack of suitable potency data may lead to situations where bridging clinical efficacy data are required to solve the problems of the potency testing, for example where comparability of different clinical batches is unclear. In this article the challenges of potency testing are discussed together with examples of assays used for different CGTs/ATMPs and the available guidance addressing differences between the European Union and the United States.

Chronic non healing wounds in diabetic patients still impose a major problem in modern medicine. Especially additional peripheral vascular disease complicates treatment success in these patients. Thus, we analyzed the effects of 11,12 epoxyeicosatrienoic acid (EET) in a combined model of hyperglycemia and ischemia in mice. Hyperglycemia was induced by Streptozotozin 2 weeks prior to wounding. 3 days before wound creation 2 of the 3 suppling vessels of the moue ear were cautherized for ischemia. Either 11,12 EET or solvent for control was applied. Wound closure as well as TNF-α, TGF-β, SDF-1α, VEGF, CD31, and Ki67 were measured. The wounds closed on day 14.4 ± 0.4 standard deviation (SD). 11,12 EET treatment enhanced healing to 9.8 ± 0.6 SD. TNF-α level was augmented on day 9 compared to control and receded on day 18. TGF-β seemed to be elevated all days observed after 11,12 EET treatment. SDF-1α was enhanced on day 6 and 9 by 11,12 EET, and VEGF on day 6 and 18 as well as CD13 on day 3, 6, and 18. 11,12 EET did not alter Ki67. 11,12 EET are able to rescue deteriorated wound healing in a combined model of hyperglycamia and ischemia by resolution of inflammation, augmentation of neovascularization and increasing expression of TGF-β as well as SDF-1α.

Wounds in diabetic patients, especially diabetic foot ulcers, are more difficult to heal compared with normal wounds and can easily deteriorate, leading to amputation. Common treatments cannot heal diabetic wounds or control their many complications. Growth factors are found to play important roles in regulating complex diabetic wound healing. Different growth factors such as transforming growth factor beta 1, insulin-like growth factor, and vascular endothelial growth factor play different roles in diabetic wound healing. This implies that a therapeutic modality modulating different growth factors to suit wound healing can significantly improve the treatment of diabetic wounds. Further, some current treatments have been shown to promote the healing of diabetic wounds by modulating specific growth factors. The purpose of this study was to discuss the role played by each growth factor in therapeutic approaches so as to stimulate further therapeutic thinking.

Vascular endothelial growth factor A (VEGF-A) has therapeutic cardiovascular effects, but delivery challenges have impeded clinical development. We report the first clinical study of naked mRNA encoding VEGF-A (AZD8601) injected into the human heart. EPICCURE (ClinicalTrials.gov: NCT03370887) was a randomized, double-blind study of AZD8601 in patients with left ventricular ejection fraction (LVEF) 30%-50% who were undergoing elective coronary artery bypass surgery. Thirty epicardial injections of AZD8601 (total 3 mg) or placebo in citrate-buffered saline were targeted to ischemic but viable myocardial regions mapped using quantitative [15O]-water positron emission tomography. Seven patients received AZD8601 and four received placebo and were followed for 6 months. There were no deaths or treatment-related serious adverse events and no AZD8601-associated infections, immune reactions, or arrhythmias. Exploratory outcomes indicated potential improvement in LVEF, Kansas City Cardiomyopathy Questionnaire scores, and N-terminal pro-B-type natriuretic peptide levels, but the study is limited in size, and significant efficacy conclusions are not possible from the dataset. Naked mRNA without lipid encapsulation may provide a safe delivery platform for introducing genetic material to cardiac muscle, but further studies are needed to confirm efficacy and safety in a larger patient pool.

Experiments confirmed that circular RNAs contributed to the pathogenesis of diabetic foot ulcers (DFUs). CircHIPK3 was upregulated in type 2 diabetes mellitus (T2DM), but its role in DFU remained unknown. Our study aimed to investigate the regulatory functions of exosomal circHIPK3 and its potential mechanisms in DFU.

Exosomal size and distribution, marker proteins, and circHIPK3 levels were evaluated by transmission electron microscope, ExoView R200, western blot, and qRT-PCR. Flow cytometry, MTT, Wound healing assays, and tube formation assays were used to assess the roles of exosomal circHIPK3 in high glucose (HG)-treated human umbilical vein endothelial cells (HUVECs). The relationships between Nrf2/VEGFA/circHIPK3 and miR-20b-5p, and between Nrf2 and VEGFA were determined by luciferase reporter assay and RNA immunoprecipitation. We used cell and mice models to investigate the mechanisms of exosomal circHIPK3 under diabetic conditions.

CircHIPK3 was significantly upregulated in exo-circHIPK3 rather than exo-vector. Exo-circHIPK3 remarkably inhibited cell apoptosis but promoted cell proliferation, migration, and tube formation in HG-treated HUVECs. Luciferase reporter and RIP assays showed that miR-20b-5p targeted and inhibited Nrf2 and VEGFA, and circHIPK3 acted as a ceRNA of miR-20b-5p to inhibit the binding to its downstream genes Nrf2 and VEGFA. Mechanistically, circHIPK3 promoted cell proliferation, migration, and angiogenesis via downregulating miR-20b-5p to upregulate Nrf2 and VEGFA. However, the overexpressed miR-20b-5p could abolish the promoting effects of circHIPK3 overexpression on cell proliferation, migration, and tube formation under HG conditions.

UCMSCs-derived exosomal circHIPK3 protected HG-treated HUVECs via miR-20b-5p/Nrf2/VEGFA axis. The exosomal circHIPK3 might be a therapeutic candidate to treat DFU.

Although the currently available pharmacological assays can cure most pathological disorders, they have limited therapeutic value in relieving certain disorders like myocardial infarct, peripheral vascular disease, amputated limbs, or organ failure (e.g. renal failure). Pilot studies to overcome such problems using regenerative medicine (RM) delivered promising data. Comprehensive investigations of RM in zebrafish or reptilians are necessary for better understanding. However, the precise mechanisms remain poorly understood despite the tremendous amount of data obtained using the zebrafish model investigating the exact mechanisms behind their regenerative capability. Indeed, understanding such mechanisms and their application to humans can save millions of lives from dying due to potentially life-threatening events. Recent studies have launched a revolution in replacing damaged human organs via different approaches in the last few decades. The newly established branch of medicine (known as Regenerative Medicine aims to enhance natural repair mechanisms. This can be done through the application of several advanced broad-spectrum technologies such as organ transplantation, tissue engineering, and application of Scaffolds technology (support vascularization using an extracellular matrix), stem cell therapy, miRNA treatment, development of 3D mini-organs (organoids), and the construction of artificial tissues using nanomedicine and 3D bio-printers. Moreover, in the next few decades, revolutionary approaches in regenerative medicine will be applied based on artificial intelligence and wireless data exchange, soft intelligence biomaterials, nanorobotics, and even living robotics capable of self-repair. The present work presents a comprehensive overview that summarizes the new and future advances in the field of RM.

Protein replacement therapy is an umbrella term used for medical treatments that aim to substitute or replenish specific protein deficiencies that result either from the protein being absent or non-functional due to mutations in affected patients. Traditionally, such an approach requires a well characterized but arduous and expensive protein production procedure that employs in vitro expression and translation of the pharmaceutical protein in host cells, followed by extensive purification steps. In the wake of the SARS-CoV-2 pandemic, mRNA-based pharmaceuticals were recruited to achieve rapid in vivo production of antigens, proving that the in vivo translation of exogenously administered mRNA is nowadays a viable therapeutic option. In addition, the urgency of the situation and worldwide demand for mRNA-based medicine has led to an evolution in relevant technologies, such as in vitro transcription and nanolipid carriers. In this review, we present preclinical and clinical applications of mRNA as a tool for protein replacement therapy, alongside with information pertaining to the manufacture of modified mRNA through in vitro transcription, carriers employed for its intracellular delivery and critical quality attributes pertaining to the finished product.

Scars caused by skin injuries after burns, wounds, abrasions and operations have serious physical and psychological effects on patients. In recent years, the research of scar free wound repair has been greatly expanded. However, understanding the complex mechanisms of wound healing, in which various cells, cytokines and mechanical force interact, is critical to developing a treatment that can achieve scarless wound healing. Therefore, this paper reviews the types of wounds, the mechanism of scar formation in the healing process, and the current research progress on the dual consideration of wound healing and scar prevention, and some strategies for the treatment of scar free wound repair.

Endothelial cells play critical roles in circulatory homeostasis and are also the gateway to the major organs of the body. Dysfunction, injury, and gene expression profiles of these cells can cause, or are caused by, prevalent chronic diseases such as diabetes, cardiovascular disease, and cancer. Modulation of gene expression within endothelial cells could therefore be therapeutically strategic in treating longstanding disease challenges. Lipid nanoparticles (LNP) have emerged as potent, scalable, and tunable carrier systems for delivering nucleic acids, making them attractive vehicles for gene delivery to endothelial cells. Here, we discuss the functions of endothelial cells and highlight some receptors that are upregulated during health and disease. Examples and applications of DNA, mRNA, circRNA, saRNA, siRNA, shRNA, miRNA, and ASO delivery to endothelial cells and their targets are reviewed, as well as LNP composition and morphology, formulation strategies, target proteins, and biomechanical factors that modulate endothelial cell targeting. Finally, we discuss FDA-approved LNPs as well as LNPs that have been tested in clinical trials and their challenges, and provide some perspectives as to how to surmount those challenges.

Cardiovascular diseases (CVD) remain a substantial global health problem and the leading cause of death worldwide. Although many conventional small-molecule treatments are available to support the cardiac function of the patient with CVD, they are not effective as a cure. Among potential targets for gene therapy are severe cardiac and peripheral ischemia, heart failure, vein graft failure, and some forms of dyslipidemias. In the last three decades, multiple gene therapy tools have been used for heart diseases caused by proteins, plasmids, adenovirus, and adeno-associated viruses (AAV), but these remain as unmet clinical needs. These gene therapy methods are ineffective due to poor and uncontrolled gene expression, low stability, immunogenicity, and transfection efficiency. The synthetic modified mRNA (modRNA) presents a novel gene therapy approach which provides a transient, stable, safe, non-immunogenic, controlled mRNA delivery to the heart tissue without any risk of genomic integration, and achieves a therapeutic effect in different organs, including the heart. The mRNA translation starts in minutes, and remains stable for 8-10 days (pulse-like kinetics). The pulse-like expression of modRNA in the heart induces cardiac repair, cardiomyocyte proliferation and survival, and inhibits cardiomyocyte apoptosis post-myocardial infarction (MI). Cell-specific (cardiomyocyte) modRNA translation developments established cell-specific modRNA therapeutics for heart diseases. With these laudable characteristics, combined with its expression kinetics in the heart, modRNA has become an attractive therapeutic for the treatment of CVD. This review discusses new developments in modRNA therapy for heart diseases.

Over the last seven decades, a significant scientific contribution took place in the delineation of the implications of vascular endothelial-derived growth factor (VEGF) in the processes of angiogenesis. Under pathological conditions, mainly in response to hypoxia or ischemia, elevated VEGF levels promote vascular damage and the growth of abnormal blood vessels. Indeed, the development of VEGF biology has revolutionized our understanding of its role in pathological conditions. Hence, targeting VEGF or VEGF-mediated molecular pathways could be an excellent therapeutic strategy for managing cancers and intraocular neovascular disorders. Although anti-VEGF therapies, such as monoclonal antibodies and small-molecule tyrosine kinase inhibitors, have limited clinical efficacy, they can still significantly improve the overall survival rate. This thus demands further investigation through the development of alternative strategies in the management of VEGF-mediated pathological angiogenesis. This review article focuses on the recent developments toward the delineation of the functional biology of VEGF and the role of anti-VEGF strategies in the management of tumor and eye pathologies. Moreover, therapeutic angiogenesis, an exciting frontier for the treatment of ischemic disorders, is highlighted in this review, including wound healing.

Fibroblasts are the main cells of connective tissue and have pivotal roles in the proliferative and maturation phases of wound healing. These cells can secrete various cytokines, growth factors, and collagen. Vascular endothelial growth factor (VEGF) is a unique factor in the migration process of fibroblast cells through induces wound healing cascade components such as angiogenesis, collagen deposition, and epithelialization. This study aimed to create VEGF₁₆₅ overexpressing fibroblast cells to evaluate angiogenesis function in wound healing. In vitro, a novel recombinant expression vector, pcDNA3.1(-)-VEGF, was produced and transfected into the fibroblast cells. Following selecting fibroblast cells with hygromycin, recombinant cells were investigated in terms of VEGF expression by quantifying and qualifying methods. Mechanical, physical, and survival properties of polyurethane-cellulose acetate (PU-CA) scaffold were investigated. Finally, in vivo, the angiogenic potential was evaluated in four groups containing control, PU-CA, PU-CA with fibroblast cells, and VEGF-expressing cells on days 0, 2, 5, 12 and 15. Wound biopsies were harvested and the healing process was histopathologically evaluated on different days. qRT-PCR showed VEGF overexpression (sevenfold) in genetically-manipulated cells compared to fibroblast cells. Recombinant VEGF expression was also confirmed by western blotting. Manipulated fibroblast cells represented more angiogenesis than other groups on the second day after surgery, which was also confirmed by the antiCD31 antibody. The percentage of wound closure area on day 5 in genetically-manipulated Hu02 and Hu02 groups showed a significant reduction of wound area compared to other groups. These findings indicate that overexpression of VEGF₁₆₅ in fibroblast cells results in enhanced angiogenesis and formation of granulated tissue in the early stage of the healing process, which can show its therapeutic potential in patients with impaired wound healing and also provide functional support for gene therapy.

The extraordinary success of mRNA vaccines against coronavirus disease 2019 (COVID-19) has renewed interest in mRNA as a means of delivering therapeutic proteins. Early clinical trials of mRNA therapeutics include studies of paracrine vascular endothelial growth factor (VEGF) mRNA for heart failure and of CRISPR-Cas9 mRNA for a congenital liver-specific storage disease. However, a series of challenges remains to be addressed before mRNA can be established as a general therapeutic modality with broad relevance to both rare and common diseases. An array of new technologies is being developed to surmount these challenges, including approaches to optimize mRNA cargos, lipid carriers with inherent tissue tropism and in vivo percutaneous delivery systems. The judicious integration of these advances may unlock the promise of biologically targeted mRNA therapeutics, beyond vaccines and other immunostimulatory agents, for the treatment of diverse clinical indications.

Atherosclerosis is the main underlying pathology for many cardiovascular diseases (CVDs), which are the leading cause of death globally and represent a serious health crisis. Atherosclerosis is a chronic condition that can lead to myocardial infarction, ischemic cardiomyopathy, stroke, and peripheral arterial disease. Elevated plasma lipids, hypertension, and high glucose are the major risk factors for developing atherosclerotic plaques. To date, most pharmacological therapies aim to control these risk factors, but they do not target the plaque-causing cells themselves. In patients with acute coronary syndromes, surgical revascularization with percutaneous coronary intervention has greatly reduced mortality rates. However, stent thrombosis and neo-atherosclerosis have emerged as major safety concerns of drug eluting stents due to delayed re-endothelialization. This review summarizes the major milestones, strengths, and limitations of current anti-atherosclerotic therapies. It provides an overview of the recent discoveries and emerging game-changing technologies in the fields of nanomedicine, mRNA therapeutics, and gene editing that have the potential to revolutionize CVD clinical practice by steering it toward precision medicine.

Diabetes mellitus is a chronic metabolic disorder commonly encountered in orthopedic patients. Both type 1 and type 2 diabetes mellitus increase fracture risk and impair fracture healing. This review examines complex etiology of impaired fracture healing in diabetes.

Recent findings point to several mechanisms leading to orthopedic complications in diabetes. Hyperglycemia and chronic inflammation lead to increased formation of advanced glycation end products and generation of reactive oxygen species, which in turn contribute to the disruption in osteoblast and osteoclast balance leading to decreased bone formation and heightening the risk of nonunion or delayed union as well as impaired fracture healing. The mechanisms attributing to this imbalance is secondary to an increase in pro-inflammatory mediators leading to premature resorption of callus cartilage and impaired bone formation due to compromised osteoblast differentiation and their apoptosis. Other mechanisms include disruption in the bone's microenvironment supporting different stages of healing process including hematoma and callus formation, and their resolution during bone remodeling phase. Complications of diabetes including peripheral neuropathy and peripheral vascular disease also contribute to the impairment of fracture healing. Certain diabetic drugs may have adverse effects on fracture healing. The pathophysiology of impaired fracture healing in diabetic patients is complex. This review provides an update of the most recent findings on how key mediators of bone healing are affected in diabetes.

Sepsis is caused by dysregulated host inflammatory response to infection. During sepsis, early identification and monitoring of vascular leakage are pivotal for improved diagnosis, treatment, and prognosis. However, there is a lack of research on noninvasive observation of inflammation-related vascular leakage. Here, we investigate the use of photoacoustic microscopy (PAM) for in vivo visualization of lipopolysaccharide (LPS)-induced ear vascular leakage in mice using Evans blue (EB) as an indicator. A model combining needle pricking on the mouse ear, topical smearing of LPS on the mouse ear, and intravenous tail injection of EB is developed. Topical application of LPS is expected to induce local vascular leakage in skin. Inflammatory response is first validated by ex vivo histology and enzyme-linked immunosorbent assay. Then, local ear vascular leakage is confirmed by ex vivo measurement of swelling, thickening, and EB leakage. Finally, PAM for in vivo identification and evaluation of early vascular leakage using the model is demonstrated. For PAM, common excitation wavelength of 532 nm is used, and an algorithm is developed to extract quantitative metrics for EB leakage. The results show potential of PAM for noninvasive longitudinal monitoring of peripheral skin vascular leakage, which holds promise for clinical sepsis diagnosis and management.