Chitosan Particles Complexed with CA5-HIF-1α Plasmids Increase Angiogenesis and Improve Wound Healing.

Wound therapies involving gene delivery to the skin have significant potential due to the advantage and ease of local treatment. However, choosing the appropriate vector to enable successful gene expression while also ensuring that the treatment's immediate material components are conducive to healing itself is critical. In this study, we utilized a particulate formulation of the polymer chitosan (chitosan particles, CPs) as a non-viral vector for the delivery of a plasmid encoding human CA5-HIF-1α, a degradation resistant form of HIF-1α, to enhance wound healing. We also compared the angiogenic potential of our treatment (HIF/CPs) to that of chitosan particles containing only the plasmid backbone (bb/CPs) and the chitosan particle vector alone (CPs). Our results indicate that chitosan particles exert angiogenic effects that are enhanced with the human CA5-HIF-1α-encoded plasmid. Moreover, HIF/CPs enhanced wound healing in diabetic db/db mice (p < 0.01), and healed tissue was found to contain a significantly increased number of blood vessels compared to bb/CPs (p < 0.01), CPs (p < 0.05) and no-treatment groups (p < 0.01). Thus, this study represents a method of gene delivery to the skin that utilizes an inherently pro-wound-healing polymer as a vector for plasmid DNA that has broad application for the expression of other therapeutic genes.

Potential Role of Silencing Ribonucleic Acid for Esophageal Cancer Treatment.

INTRODUCTION: Esophageal cancer is an aggressive malignancy with high mortality. Optimal treatment of esophageal cancer remains an elusive goal. Ribonucleic acid (RNA) interference is a novel potential targeted approach to treat esophageal cancer. Targeting oncogenes that can alter critical cellular functions with silencing RNA molecules is a promising approach. The silencing of specific oncogenes in esophageal cancer cells in the experimental setting has been shown to decrease the expression of oncogenic proteins. This has resulted in cell apoptosis, reduction in cell proliferation, reduced invasion, migration, epithelial-mesenchymal transition, decrease in tumor angiogenesis and metastasis, and overcoming drug resistance. The Hedgehog (Hh) signaling pathway has been shown to be involved in esophageal adenocarcinoma formation in a reflux animal model. In addition to Hh, we will focus on other targets with clinical potential in the treatment of esophageal cancer. MATERIALS AND METHODS: We searched for articles published from 2005 to August 2020 that studied the siRNA effects on inhibiting esophageal cancer formation in experimental settings. We used combinations of the following terms for searching: "esophageal cancer," "RNA interference," "small interfering RNA," "siRNA," "silencing RNA," "Smoothened (Smo)," "Gli," "Bcl-2," "Bcl-XL," "Bcl-W,″ "Mcl-1," "Bfl-1," "STAT3,"and "Hypoxia inducible factor (HIF)". A total of 21 relevant articles were found. RESULTS AND CONCLUSIONS: Several proto-oncogenes/oncogenes including Hh pathway mediators, glioma-associated oncogene homolog 1 (Gli-1), Smoothened (Smo), and antiapoptotic Bcl-2 have potential as targets for silencing RNA in the treatment of esophageal cancer.

Preconditioning of surgical pedicle flaps with DNA plasmid expressing hypoxia-inducible factor-1α (HIF-1α) promotes tissue viability.

Ischemic necrosis of surgical flaps after reconstruction is a major clinical problem. Hypoxia-inducible factor-1α (HIF-1α) is considered the master regulator of the adaptive response to hypoxia. Among its many properties, it regulates the expression of genes encoding angiogenic growth factors, which have a short half-life in vivo. To achieve a continuous application of the therapeutic, we utilized DNA plasmid delivery. Transcription of the DNA plasmid confirmed by qRT-PCR showed significantly increased mRNA for HIF-1α in the transfected tissue compared to saline control tissue. Rats were preconditioned by injecting with either HIF-1α DNA plasmid or saline intradermally in the designated flap region on each flank. Seven days after preconditioning, each rat had two isolated pedicle flaps raised with a sterile silicone sheet implanted between the skin flap and muscle layer. The flaps preconditioned with HIF-1α DNA plasmid had significantly less necrotic area. Angiogenesis measured by CD31 staining showed a significant increase in the number of vessels per high powered field in the HIF-1α group (p < 0.05). Our findings offer a potential therapeutic strategy for significantly promoting the viability of surgical pedicle flaps by ischemic preconditioning with HIF-1α DNA plasmid.

Targeting the Hedgehog Pathway Using Itraconazole to Prevent Progression of Barrett's Esophagus to Invasive Esophageal Adenocarcinoma.

OBJECTIVE: The aim of the study was to investigate whether inhibition of Sonic Hedgehog (SHH) pathway would prevent progression of Barrett's Esophagus (BE) to esophageal adenocarcinoma. BACKGROUND: The hedgehog signaling pathway is a leading candidate as a molecular mediator of BE and esophageal adenocarcinoma (EAC). Repurposed use of existing off-patent, safe and tolerable drugs that can inhibit hedgehog, such as itraconazole, could prevent progression of BE to EAC. METHODS: The efficacy of itraconazole was investigated using a surgical rat reflux model of Barrett's Metaplasia (BM). Weekly intraperitoneal injections of saline (control group) or itraconazole (treatment group; 200 mg/kg) were started at 24 weeks postsurgery. Esophageal tissue was harvested at 40 weeks. The role of the Hh pathway was also evaluated clinically. Esophageal tissue was harvested after 40 weeks for pathological examination and evaluation of the SHH pathway by immunohistochemistry. RESULTS: BM was present in control animals 29 of 31 (93%) versus itraconazole 22 of 24 (91%). EAC was significantly lower in itraconazole 2 of 24 (8%) versus control 10 of 31 (32%), respectively (P = 0.033). Esophageal SHH levels were lower in itraconazole vs control (P = 0.12). In esophageal tissue from humans with recurrent or persistent dysplastic BE within 24 months of ablative treatment, strong SHH and Indian Hedgehog expression occurred in distal BE versus proximal squamous epithelium, odds ratio = 6.1 (95% confidence interval: 1.6, 23.4) and odds ratio = 6.4 (95% confidence interval: 1.2, 32.8), respectively. CONCLUSION: Itraconazole significantly decreases EAC development and SHH expression in a preclinical animal model of BM. In humans, BE tissue expresses higher SHH, Indian Hedgehog, and bone morphogenic protein levels than normal squamous esophageal epithelium.

Strengthening the skin with topical delivery of keratinocyte growth factor-1 using a novel DNA plasmid.

Fragile skin, susceptible to decubitus ulcers and incidental trauma, is a problem particularly for the elderly and for those with spinal cord injury. Here, we present a simple approach to strengthen the skin by the topical delivery of keratinocyte growth factor-1 (KGF-1) DNA. In initial feasibility studies with the novel minimalized, antibiotic-free DNA expression vector, NTC8385-VA1, the reporter genes luciferase and enhanced green fluorescent protein were delivered. Transfection was documented when luciferase expression significantly increased after transfection. Microscopic imaging of enhanced green fluorescent protein-transfected skin showed green fluorescence in hair follicles, hair shafts, and dermal and superficial epithelial cells. With KGF-1 transfection, KGF-1 mRNA level and protein production were documented with quantitative reverse transcriptase-polymerase chain reaction and immunohistochemistry, respectively. Epithelial thickness of the transfected skin in the KGF group was significantly increased compared with the control vector group (26 ± 2 versus 16 ± 4 µm) at 48 hours (P = 0.045). Dermal thickness tended to be increased in the KGF group (255 ± 36 versus 162 ± 16 µm) at 120 hours (P = 0.057). Biomechanical assessment showed that the KGF-1-treated skin was significantly stronger than control vector-transfected skin. These findings indicate that topically delivered KGF-1 DNA plasmid can increase epithelial thickness and strength, demonstrating the potential of this approach to restore compromised skin.

HOTAIR-Loaded Mesenchymal Stem/Stromal Cell Extracellular Vesicles Enhance Angiogenesis and Wound Healing.

Chronic wounds remain a substantial source of morbidity worldwide. An emergent approach that may be well-suited to induce the complex, multicellular processes such as angiogenesis that are required for wound repair is the use of extracellular vesicles (EVs). EVs contain a wide variety of proteins and nucleic acids that enable multifactorial signaling. Here, the capability of EVs is leveraged to be engineered via producer cell modification to investigate the therapeutic potential of EVs from mesenchymal stem/stromal cells (MSCs) transfected to overexpress long non-coding RNA HOX transcript antisense RNA (HOTAIR). HOTAIR is previously shown by the authors' group to be critical in mediating angiogenic effects of endothelial cell EVs, and MSCs are chosen as EV producer cells for this study due to their widely reported intrinsic angiogenic properties. The results indicate that MSCs overexpressing HOTAIR (HOTAIR-MSCs) produce EVs with increased HOTAIR content that promote angiogenesis and wound healing in diabetic (db/db) mice. Further, endothelial cells exposed to HOTAIR-MSC EVs exhibit increased HOTAIR content correlated with upregulation of the angiogenic protein vascular endothelial growth factor. Thus, this study supports EV-mediated HOTAIR delivery as a strategy for further exploration toward healing of chronic wounds.

High-speed living polymerization of polar vinyl monomers by self-healing silylium catalysts.

This contribution describes the development and demonstration of the ambient-temperature, high-speed living polymerization of polar vinyl monomers (M) with a low silylium catalyst loading (≤ 0.05 mol % relative to M). The catalyst is generated in situ by protonation of a trialkylsilyl ketene acetal ((R)SKA) initiator (I) with a strong Brønsted acid. The living character of the polymerization system has been demonstrated by several key lines of evidence, including the observed linear growth of the chain length as a function of monomer conversion at a given [M]/[I] ratio, near-precise polymer number-average molecular weight (M(n), controlled by the [M]/[I] ratio) with narrow molecular weight distributions (MWD), absence of an induction period and chain-termination reactions (as revealed by kinetics), readily achievable chain extension, and the successful synthesis of well-defined block copolymers. Fundamental steps of activation, initiation, propagation, and catalyst "self-repair" involved in this living polymerization system have been elucidated, chiefly featuring a propagation "catalysis" cycle consisting of a rate-limiting C--C bond formation step and fast release of the silylium catalyst to the incoming monomer. Effects of acid activator, catalyst and monomer structure, and reaction temperature on polymerization characteristics have also been examined. Among the three strong acids incorporating a weakly coordinating borate or a chiral disulfonimide anion, the oxonium acid [H(Et(2)O)(2)](+)[B(C(6)F(5))(4)](-) is the most effective activator, which spontaneously delivers the most active R(3)Si(+), reaching a high catalyst turn-over frequency (TOF) of 6.0×10(3) h(-1) for methyl methacrylate polymerization by Me(3)Si(+) or an exceptionally high TOF of 2.4×10(5) h(-1) for n-butyl acrylate polymerization by iBu(3)Si(+), in addition to its high (>90 %) to quantitative efficiencies and a high degree of control over M(n) and MWD (1.07-1.12). An intriguing catalyst "self-repair" feature has also been demonstrated for the current living polymerization system.

A powerful chiral counteranion motif for asymmetric catalysis.

Room to swing a cat: A chiral disulfonimide has been designed as a powerful new motif for asymmetric catalysis. As a first illustration, a highly efficient and enantioselective Mukaiyama aldol reaction has been developed (see scheme). The actual catalyst is proposed to be an N-silyl imide which is generated in situ.

Efficacy of Chitosan-Based Dressing for Control of Bleeding in Excisional Wounds.

Introduction: Excessive bleeding is a complication of wound debridement in patients receiving anticoagulation treatment. Chitosan is a linear, positively charged polysaccharide that has potential as a hemostatic topical dressing. This study examined the hemostatic efficacy of the chitosan based Opticell dressing (Medline Industries, Chicago, Ill) in heparinized rats with excisional wounds mimicking debridement. Methods: Three paired 12-mm excisional wounds were created on the dorsum of 600-g Sprague-Dawley rats 2 hours after intraperitoneal injection of heparin 800 IU/kg. Opticell or gauze dressings were applied with 3 seconds of gentle pressure. Results:Total Bleeding: The dressings were left in place until cessation of bleeding. Ten minutes was enough time for complete bleeding cessation in both groups. Gauze and Opticell were weighed before and after bleeding cessation, with the difference representing blood loss. Total blood loss was 627 ± 47 mg/10 min with the standard gauze, but 247 ± 47 mg/10 min with Opticell (P = .002 Mann-Whitney). N = 6 wounds per group. Rate of Bleeding: Gauze and Opticell dressings were removed and instantly replaced with 3 seconds of gentle pressure every minute until bleeding cessation. The removed dressings were weighed before and after application. There was less bleeding in the Opticell group at minutes 1, 2, and 3. Gauze: 183 ± 40, 140 ± 30, and 109 ± 15 mg/min vs Opticell: 91 ± 17, 54 ± 8, and 57 ± 11 mg/min). Analysis of variance, Tukey's test, P < .05. N = 12 wounds per group. Conclusion: Topical application of Opticell dressing with chitosan has hemostatic effects that could be a useful tool to control bleeding associated with wound debridement.

Topical Reformulation of Valsartan for Treatment of Chronic Diabetic Wounds.

Chronic wounds are among the most devastating and difficult to treat consequences of diabetes. Dysregulation of the skin renin-angiotensin system is implicated in abnormal wound healing in diabetic and older adults. Given this, we sought to determine the effects of topical reformulations of the angiotensin type 1 receptor blockers losartan and valsartan and the angiotensin-converting enzyme inhibitor captopril on wound healing in diabetic and aged mice with further validation in older diabetic pigs. The application of 1% valsartan gel compared with other tested formulations and placebo facilitated and significantly accelerated closure time and increased tensile strength in mice, and was validated in the porcine model. One percent of valsartan gel-treated wounds also exhibited higher mitochondrial content, collagen deposition, phosphorylated mothers against decapentaplegic homologs 2 and 3 and common mothers against decapentaplegic homolog 4, alpha-smooth muscle actin, CD31, phospho-vascular endothelial growth factor receptor 2, and p42/44 mitogen-activated protein kinase. Knockout of the angiotensin subtype 2 receptors abolished the beneficial effects of angiotensin type 1 receptor blockers, suggesting a role for angiotensin subtype 2 receptors in chronic wound healing.

Cellular GFP Toxicity and Immunogenicity: Potential Confounders in in Vivo Cell Tracking Experiments.

Green Fluorescent protein (GFP), used as a cellular tag, provides researchers with a valuable method of measuring gene expression and cell tracking. However, there is evidence to suggest that the immunogenicity and cytotoxicity of GFP potentially confounds the interpretation of in vivo experimental data. Studies have shown that GFP expression can deteriorate over time as GFP tagged cells are prone to death. Therefore, the cells that were originally marked with GFP do not survive and cannot be accurately traced over time. This review will present current evidence for the immunogenicity and cytotoxicity of GFP in in vivo studies by characterizing these responses.

Knockout of Angiotensin AT2 receptors accelerates healing but impairs quality.

Wounds are among the most common, painful, debilitating and costly conditions in older adults. Disruption of the angiotensin type 1 receptors (AT1R), has been associated with impaired wound healing, suggesting a critical role for AT1R in this repair process. Biological functions of angiotensin type 2 receptors (AT2R) are less studied. We investigated effects of genetically disrupting AT2R on rate and quality of wound healing. Our results suggest that AT2R effects on rate of wound closure depends on the phase of wound healing. We observed delayed healing during early phase of wound healing (inflammation). An accelerated healing rate was seen during later stages (proliferation and remodeling). By day 12, fifty percent of AT2R(-/-) mice had complete wound closure as compared to none in either C57/BL6 or AT1R(-/-) mice. There was a significant increase in AT1R, TGFß1 and TGFß2 expression during the proliferative and remodeling phases in AT2R(-/-) mice. Despite the accelerated closure rate, AT2R(-/-) mice had more fragile healed skin. Our results suggest that in the absence of AT2R, wound healing rate is accelerated, but yielded worse skin quality. Elucidating the contribution of both of the angiotensin receptors may help fine tune future intervention aimed at wound repair in older individuals.

Tie2-dependent knockout of HIF-1 impairs burn wound vascularization and homing of bone marrow-derived angiogenic cells.

AIMS: Hypoxia-inducible factor 1 (HIF-1) is a heterodimer composed of HIF-1α and HIF-1ß subunits. HIF-1 is known to promote tissue vascularization by activating the transcription of genes encoding angiogenic factors, which bind to receptors on endothelial cells (ECs) and bone marrow-derived angiogenic cells (BMDACs). In this study, we analysed whether HIF-1 activity in the responding ECs and BMDACs is also required for cutaneous vascularization during burn wound healing. METHODS AND RESULTS: We generated mice with floxed alleles at the Hif1a or Arnt locus encoding HIF-1α and HIF-1ß, respectively. Expression of Cre recombinase was driven by the Tie2 gene promoter, which is expressed in ECs and bone marrow cells. Tie2Cre(+) and Tie2Cre(-) mice were subjected to burn wounds of reproducible diameter and depth. Deficiency of HIF-1α or HIF-1ß in Tie2-lineage cells resulted in delayed wound closure, reduced vascularization, decreased cutaneous blood flow, impaired BMDAC mobilization, and decreased BMDAC homing to burn wounds. CONCLUSION: HIF-1 activity in Tie2-lineage cells is required for the mobilization and homing of BMDACs to cutaneous burn wounds and for the vascularization of burn wound tissue.

Subterranean termite prophylactic secretions and external antifungal defenses.

Termites exploit environments that make them susceptible to infection and rapid disease transmission. Gram-negative bacteria binding proteins (GNBPs) signal the presence of microbes and in some insects directly damage fungal pathogens with ß-1,3-glucanase activity. The subterranean termites Reticulitermes flavipes and Reticulitermes virginicus encounter soil entomopathogenic fungi such as Metarhizium anisopliae, which can evade host immune responses after penetrating the cuticle. An external defense that prevents invasion of fungal pathogens could be crucial in termites, allowing them to thrive under high pathogenic pressures. We investigated the role of secreted ß-1,3-glucanases in Reticulitermes defenses against M. anisopliae. Our results show that these termites secrete antifungal ß-1,3-glucanases on the cuticle, and the specific inhibition of GNBP associated ß-1,3-glucanase activity with d-δ-gluconolactone (GDL) reduces this activity and can cause significant increases in mortality after exposure to M. anisopliae. Secreted ß-1,3-glucanases appear to be essential in preventing infection by breaking down fungi externally.

Synthesis and biological evaluation of cruentaren A analogues.

The complex macrolide cruentaren A is a highly selective and potent inhibitor of F-ATPase (F-type adenosine triphosphatase). As it shows some resemblance to benzolactone enamides like apicularen A, it was of interest to perform some structure-activity studies to delineate the key functional groups that are responsible for the activity. Building upon our previously developed route to cruentaren A, which is based on a ring-closing alkyne metathesis reaction (RCAM), several cruentaren analogues were prepared. Replacement of the 3-hydroxy hexanoic part with acids that lack the hydroxy group function resulted in a significant drop in cytotoxicity and F-ATPase inhibition. Furthermore, two enamide analogues 23 and 50 were synthesized. However, these compounds were only cytotoxic in the micromolar range. Under the conditions for cleavage of the C3 aromatic methyl ether, the enamide function was transformed to the corresponding oxazinanone, resulting in analogues 25 and 52.

Perioperative complications of laparoscopic radical prostatectomy: the Montsouris 3-year experience.

PURPOSE: We prospectively evaluated the morbidity, and minor and major complications of laparoscopic radical prostatectomy performed by a single surgical team. MATERIALS AND METHODS: Between January 28, 1998 and February 28, 2001, 567 patients 42 to 77 years old (mean age plus or minus standard deviation 63.5 +/- 6) with clinically localized prostate cancer underwent laparoscopic radical prostatectomy, including 458 (80.6%), without lymphadenectomy. Mean body mass index was 25.3 +/- 2.9 (range 17.3 to 37.5). American Society of Anesthesiologists score was 1 to 3 in 65%, 27% and 8% of cases, respectively. A total of 12 patients (2.1%) had undergone intra-abdominal surgery below the mesocolon and 40 had undergone urological surgery. Intraoperative and postoperative data were recorded as well as all complications and their severity score within the initial 30 days postoperatively. RESULTS: A total of 105 complications were observed in 97 patients (17.1%), including 21 major (3.7%) and 83 minor (14.6%) complications. Of the patients 21 (3.7%) underwent reoperation for a postoperative complication, including 10 (1.76%) who required an intensive care unit stay. Seven cases (1.2%) were converted to conventional retropubic radical prostatectomy. Mean blood loss was 380 +/- 195 ml. and the overall transfusion rate was 4.9%. In 2 patients (0.3%) deep vein thrombosis was associated with another surgical complication but not with pulmonary embolism. Urological, bowel and hemorrhagic complications represented 66.6%, 16.2% and 7.6% (total 89.4%) of all complications, and 20%, 33.3% and 33.3% of all repeat interventions, respectively. CONCLUSIONS: Laparoscopic radical prostatectomy was performed according to the defined protocol with no complications in 82.9% of patients. The morbidity of this approach compares favorably with that of retropubic surgery. Growing experience and knowledge sharing concerning the prevention and early management of these complications would make possible a further decrease in the morbidity of laparoscopic radical prostatectomy.