Laser Doppler Imaging Research Articles

732 Examining Staff Views and Perceptions After Implementation of a Laser Doppler Imaging Device Into Practice

IntroductionLaser doppler imaging (LDI) has been established as an accurate diagnostic tool to assess burn depth and measure healing potential. Despite this, its use in burn centers remain limited. While studies have examined challenges to LDI use, there is a paucity of literature factoring staff views and attitudes as a barrier to implementation into burn practice. The aim of this work was to assess and examine attitudes and perspectives following implementation of an LDI protocol into acute burn injury assessment.MethodsFollowing institutional approval, a 22-question survey was disseminated among staff involved with implementation of an LDI device as a point of care tool to assess acute burn injury at a single ABA verified burn center. The survey focused on questions examining device ease of use, understanding of the underlying LDI technology, interpretation of imaging generation from wounds, and perceptions of patients’ experience. Questions were answered on a standard 5-point Likert scale. All survey data was collected anonymously into an electronic database for assessment.ResultsOverall, there were 15 respondents to the survey questions. Five respondents found the LDI device difficult to use (33%). Barriers to device ease of use included difficulty with device movement (60%), incorporation of scanning into wound care and dressing placement (60%), and management of hardware or software issues that arise during use (60%). Challenges noted by respondents external to device use was mainly high patient census (80%). Despite this, 60% of respondents found the device easier to use after performing several scans in one or more patients and 60% found that scans generated matched their assessment of burn depth. Among respondents, 66% found patients amenable to the scanning process and 80% did not feel that that the scanning process worsened patients’ pain.ConclusionsChallenges in LDI device use, implementation with wound care and dressing placement, and high patient census were identified as barriers to LDI use. Despite this, ease of device use improved with more frequent use. Identification of views and perceptions such as these can lead to protocol changes and additional training that facilitate ease of LDI use. Further examination will be required to better elucidate this information.

Human Bone Marrow Mononuclear Cells Do Not Improve Limb Perfusion in the Hindlimb Ischemia Model.

Effective treatments for chronic limb-threatening ischemia are lacking. (Pre)clinical studies on administration of bone marrow (BM) mononuclear cells (MNCs) and BM-derived mesenchymal stromal cells (MSCs) have shown variable results and no studies have directly compared administration of human BM MNCs and BM MSCs in in vivo models. We studied the effect of intramuscular administration of human BM-derived MNCs and MSCs on limb perfusion in the murine hindlimb ischemia (HLI) model. Human BM MNCs and MSCs were obtained from healthy consenting donors. Both cell types were cryopreserved before use. Twenty-four hours after induction of HLI, nude NMRI mice were randomized to receive intramuscular administration of human BM MNCs (n = 13), or BM MSCs (n = 14), or vehicle control (n = 19) in various doses. Limb perfusion was measured using laser Doppler imaging on day 0, 1, 4, 7, 10, and 14. Intramuscular injection of human BM MNCs did not improve limb perfusion as compared with vehicle over the 2 weeks after cell administration (P = 0.88, mean relative perfusion for vehicle 0.56 ± 0.04 and 0.53 ± 0.04 for BM MNCs at day 14). Administration of human BM MSCs significantly improved limb perfusion as compared with both BM MNCs and vehicle (P ≤ 0.001, mean relative perfusion at day 14 0.79 ± 0.06). Our data suggest that BM MNCs are less suitable than BM MSCs for cell-based therapy that aims to restore perfusion.

Laser doppler imaging – the role of poor burn perfusion in predicting healing time and guiding operative management

AimTo identify if the proportion of poor blood flow (blue) within an LDI (Laser doppler Imaging) image of a burn independently correlates with healing time. MethodsPatient age, gender, burn type, and burn surface area were collected from the IBID (International Burn Injury Database). All LDI images were copied from the MoorLDI2-BI- Laser Doppler (MLDI) Scanner, onto Adobe Photoshop® version 2020 for pixel counting analysis and calculation of % TBSA (Total Body Surface Area) blue. Multiple linear regression analysis determined whether a proportional relationship was present for each parameter (age, gender, % TBSA Blue and comorbidities) with healing time. Results110 patients with 197 burns were scanned with MLDI. Median age was 5 years (IQR 1–6). Median burn surface area was 1.5% (IQR 1–2.4). 56.4% of patients were male and patients were scanned an average of 2.68 days (SD±1.37) following burn injury. Number of physical comorbidities and age were found to have a statistically significant relationship with healing time (p = 0.03, p = 0.002). Gender and %TBSA blue did not have a statistically significant relationship with healing time (p = 0.07 and p = 0.058 respectively). We found a statistically significant difference in the mean healing time between burns with and without blue (3.43 weeks vs. 2.80 weeks, p = 0.0001). % TBSA Blue was more than four times higher in the operated group (0.48% vs. 0.11%) and was shown to have a statistically significant relationship with decision to operate (p = 0.027). Positive predictive value for the presence of blue on operative rate was 71.6%. Age, gender and number of comorbidities did not have a statistically significant influence on operative rate (p = 0.07, p = 0.50 and p = 0.49). Conclusion% TBSA blue was not found to be a reliable independent indicator of burn healing time, but the presence of blue within an LDI image, advanced patient age and increased number of comorbidities did have a statistically significant relationship with healing time. This suggests their standardised inclusion into management decisions regarding intermediate depth burns is warranted.

VEGF alleviates lower limb ischemia in diabetic mice by altering muscle fiber types.

Lower limb ischemia caused by diabetic foot (DF) is one of the most serious complications of diabetes. The therapeutic role of VEGF in DF is well documented. However, the mechanism for action of VEGF is still not clear. The present study aimed to explore the effects of VEGF-mediated skeletal muscle fiber type switch in angiogenesis and the treatment of DF. C57BL/6 mice housed in cages equipped with a voluntary running wheel were used to access VEGF protein level and citrate synthase activity (by ELISA) as well as muscle fiber type changes (by immunofluorescence) in the gastrocnemius muscle. C57BL/6 mice were fed on a high-fat diet for 6 weeks and then injected with streptozocin to induce diabetic lower limb ischemia model. Control adenovirus (Ad-GFP) or Ad-VEGF-GFP were then injected into the left gastrocnemius of the ischemic diabetic mice. Blood flow perfusion was examined by laser Doppler imaging at 1, 3, 7 and 14 days after adenovirus transduction. On day 14, all mice were anesthetized and sacrificed. VEGF expression levels, citrate synthase activity and muscle fiber type changes in the gastrocnemius muscle were assayed by ELISA and immunofluorescence analysis of myosin heavy chain IIa (MHCIIa) expression, respectively. Transwell assays were performed to determine whether VEGF-treated C2C12 myotubes played a role on tubule formation and migration of HUVECs. It was found that VEGF levels and citrate synthase activity were upregulated after voluntary exercise, along with the increased frequency of oxidized muscle fibers. Notably, adenovirus-mediated VEGF overexpression in the muscle also increased the frequency of oxidized (MHCIIa-positive) muscle fibers, enhanced citrate synthase activity and ameliorated lower limb ischemia in diabetic mice. VEGF treatment enhanced the phosphorylation of PI3K, Akt and AMPK (assayed by western blotting), as well as glucose consumption and metabolism (assayed by western blotting and glucose uptake assay), in the C2C12 myotubes. Interestingly, VEGF-treated C2C12 myotubes promoted the migration and tubule formation of HUVEC cells. The present findings suggest that skeletal muscle fiber conversion might be a potential approach for VEGF-mediated angiogenesis and disease treatment, which may provide new options for the prevention and treatment of DF.

E-Selectin-Overexpressing Mesenchymal Stem Cell Therapy Confers Improved Reperfusion, Repair, and Regeneration in a Murine Critical Limb Ischemia Model.

AimsNovel cell-based therapeutic angiogenic treatments for patients with critical limb ischemia may afford limb salvage. Mesenchymal stem cells (MSCs) do not overexpress E-selectin; however, we have previously demonstrated the cell-adhesion molecule's vital role in angiogenesis and wound healing. Thus, we created a viral vector to overexpress E-selectin on MSCs to increase their therapeutic profile.Methods and ResultsFemoral artery ligation induced hind limb ischemia in mice and intramuscular injections were administered of vehicle or syngeneic donor MSCs, transduced ex vivo with an adeno-associated viral vector to express either GFP+ (MSCGFP) or E-selectin-GFP+ (MSCE−selectin−GFP). Laser Doppler Imaging demonstrated significantly restored reperfusion in MSCE−selectin−GFP-treated mice vs. controls. After 3 weeks, the ischemic limbs in mice treated with MSCE−selectin−GFP had increased footpad blood vessel density, hematoxylin and eosin stain (H&E) ischemic calf muscle sections revealed mitigated muscular atrophy with restored muscle fiber size, and mice were able to run further before exhaustion. PCR array-based gene profiling analysis identified nine upregulated pro-angiogenic/pro-repair genes and downregulated Tumor necrosis factor (TNF) gene in MSCE−selectin−GFP-treated limb tissues, indicating that the therapeutic effect is likely achieved via upregulation of pro-angiogenic cytokines and downregulation of inflammation.ConclusionThis innovative cell therapy confers increased limb reperfusion, neovascularization, improved functional recovery, decreased muscle atrophy, and thus offers a potential therapeutic method for future clinical studies.

Helpful hints in deciding what and when to operate after enzymatic debridement

IntroductionIn recent years, it has become clear that the burn eschar in deep burns can be selectively removed using the enzymatically debriding agent NexoBrid® (EDNX). In deep partial-thickness burns, such selective debridement preserves all non-injured dermis, which is sometimes sufficient for spontaneous re-epithelization. Nevertheless, it can be extremely challenging to determine exactly what and when to operate after an EDNX procedure. In this manuscript, we sought to investigate the clinical aspect of the enzymatically debrided wound bed of laser Doppler imaging (LDI)-confirmed deep dermal and full-thickness burns after NexoBrid® application. This to evaluate the residual wound healing capacity and implement specific indications for surgical therapy after enzymatic debridement. Material and methodsMainly LDI-blue areas, determined between 48 h and 5d after burn and afterwards treated with EDNX were selected. Six practical and three expert EDNX users evaluated the high-quality digital images of the wound beds immediately post NexoBrid® removal and after a 2 h wet-to-dry (WTD) dressing period. ResultsOne hundred and two mainly LDI-blue areas in 32 patients were analyzed. Regarding the early decision-making, there were no significant differences in the wound bed evaluations, wound healing assessment and treatment decision of all 9 EDNX users post EDNX removal versus post WTD. Moreover, there was a good to excellent consensus between the practical and expert EDNX users in the individual wound bed evaluations. Even in the evaluation of a newly developed wound bed color code, with 7 different colors/patterns to choose from, the consensus was 80%. There was also an 84% consensus on the decision whether or not to operate.All mainly LDI-blue areas with incomplete enzymatic debridement, determined during clinical investigation by expert EDNX users, required surgery. Additionally, the expert investigators demonstrated that the following wound bed characteristics were independent predictors of the need for surgical treatment: visible fat lobules (p = 0.028), translucent fat lobules (p < 0.001), dermal step-off in the wound bed (p < 0.001), visible blood vessels (p < 0.001) and coagulated blood vessels (p = 0.023). Also, higher color code ranges on our own developed wound bed classification were significantly related to a surgical intervention (p = 0.006).When including the LDI flux values, the perfusion units were significantly different (p < 0.001) between the exclusively LDI-blue areas treated conservatively (mean 145.7) and the areas ultimately treated with autografts (mean 119.5). ConclusionTo the best of our knowledge, this study is the first to address the clinical wound bed evaluation of LDI-confirmed deep burns after NexoBrid® application. Based on our results, it is recommended to evaluate the wound bed twice: immediately after removing NexoBrid® to assess the viability of the wound bed and after the WTD period to reach a more complete decision. During these evaluations, wound bed characteristics such as incomplete debridement, visible and/or translucent fat lobules, visible and/or coagulated blood vessels and a dermal step-off in the wound bed combined with a higher range (4−5) in the newly developed wound bed color code should lead to an early and reliable decision for skin grafting. For burn centers using LDI, mean flux values below 119.5PU - in addition to the above-mentioned wound bed evaluation - are a clear indicator for surgical therapy.

Differential action of pro-angiogenic and anti-angiogenic components of Danhong injection in ischemic vascular disease or tumor models

ObjectiveWe investigate the chemical basis and mechanism of angiogenesis regulation by a multicomponent Chinese medicine Danhong injection (DHI).MethodsDHI was fractionated and screened for angiogenesis activities by in vitro tube formation and migration assays. The composition of DHI components was determined by UPLC. The effects of the main active monomers on angiogenesis-related gene and protein expression in endothelial cells were determined by qPCR and Western blotting analyses. Mouse hind limb ischemia and tumor implant models were used to verify the angiogenesis effects in vivo by Laser Doppler and bioluminescent imaging, respectively.ResultsTwo distinct chemical components, one promoting (pro-angiogenic, PAC) and the other inhibiting (anti-angiogenic, AAC) angiogenesis, were identified in DHI. PAC enhanced angiogenesis and improved recovery of ischemic limb perfusion while AAC reduced Lewis lung carcinoma growth in vivo in VEGFR-2-Luc mice. Among the PAC or AAC monomers, caffeic acid and rosmarinic acid upregulated TSP1 expression and downregulated KDR and PECAM expression. Caffeic acid and rosmarinic acid significantly decreased while protocatechuic aldehyde increased CXCR4 expression, which are consistent with their differential effects on EC migration.ConclusionsDHI is capable of bi-directional regulation of angiogenesis in disease-specific manner. The pro-angiogenesis activity of DHI promotes the repair of ischemic vascular injury, whereas the anti-angiogenesis activity inhibits tumor growth. The active pro- and anti-angiogenesis activities are composed of unique chemical combinations that differentially regulate angiogenesis-related gene networks.

Collagen triple helix repeat containing-1 promotes functional recovery of sweat glands by inducing adjacent microvascular network reconstruction in vivo.

BackgroundSweat glands (SGs) have low regenerative potential after severe burns or trauma and their regeneration or functional recovery still faces many obstacles. In practice, restoring SG function requires not only the structural integrity of the gland itself, but also its neighboring tissues, especially blood vessels. Collagen triple helix repeat containing-1 (CTHRC1) was first identified in vascular repair, and increasing reports showed a close correlation between cutaneous appendage specification, patterning and regeneration. The purpose of the present study was to clarify the role of CTHRC1 in SGs and their adjacent microvessels and find therapeutic strategies to restore SG function.MethodsThe SGs and their adjacent microvascular network of Cthrc1−/− mice were first investigated using sweat test, laser Doppler imaging, tissue clearing technique and transcriptome analysis. The effects of CTHRC1 on dermal microvascular endothelial cells (DMECs) were further explored with cell proliferation, DiI-labeled acetylated low-density lipoprotein uptake, tube formation and intercellular junction establishment assays. The effects of CTHRC1 on SG function restoration were finally confirmed by replenishing the protein into the paws of Cthrc1−/− mice.ResultsCTHRC1 is a key regulator of SG function in mice. At the tissue level, Cthrc1 deletion resulted in the disorder and reduction of the microvascular network around SGs. At the molecular level, the knockout of Cthrc1 reduced the expression of vascular development genes and functional proteins in the dermal tissues. Furthermore, CTHRC1 administration considerably enhanced SG function by inducing adjacent vascular network reconstruction.ConclusionsCTHRC1 promotes the development, morphogenesis and function execution of SGs and their neighboring vasculature. Our study provides a novel target for the restoration or regeneration of SG function in vivo.

Structural abnormalities associated with glaucoma using swept-source optical coherence tomography in patients with systemic sclerosis.

Vasospasm represents an early event in systemic sclerosis (SSc). Ocular vasospasm may induce optic nerve head (ONH) damage and has been involved in the pathogenesis of glaucoma, especially normal-tension glaucoma (NTG). We aimed to investigate the presence of structural abnormalities associated with NTG using swept-source optical coherence tomography (SS-OCT) and to correlate the OCT parameters with clinical, capillaroscopy and digital blood flow measures in patients with SSc. In this cross-sectional study, 40 patients with SSc and 23 age-matched controls were included. The following parameters were measured using SS-OCT: mean and sectoral retinal nerve fibre layer (RNFL) thickness, macular ganglion cell layer complex (GCC) thickness and ONH morphology. Nailfold capillaroscopy (NFC) and digital blood flow measurements using laser Doppler imaging (LDI) were performed in all subjects. Patients with SSc showed a thinner temporal RNFL than the controls (69.23 ± 11.74 versus 83.35 ± 20.19µm, p = 0.001). The other parameters were similar between the two groups. In SSc patients, there was an inverse correlation between the disease duration and the average, superior and inferior RNFL thickness and the GCC thickness and between Raynaud's phenomenon duration and the average RNFL and GCC thickness (p < 0.05). NFC and LDI measurements did not show correlations with OCT parameters. A thinner temporal RNFL and the correlation between Raynaud's phenomenon and disease duration and structural abnormalities on OCT suggest the presence of early ganglion cell damage in patients with SSc. Although mild, these findings indicate the need to monitor ocular abnormalities in SSc.

Current and Future Tools for Diagnosis of Kaposi's Sarcoma.

Simple SummaryKaposi’s sarcoma, a rare opportunistic tumor, is observed in four epidemiological conditions (AIDS-related, iatrogenic, endemic or classic KS). Although in most cases KS is an indolent disease, it can be locally aggressive and/or it can invade other organs than the skin, resulting in more severe presentations, especially in patients with severe immunosuppression. There is no consensus on the imaging workup that is necessary for either the initial staging of the disease or the follow-up. Future perspectives include the use of certain non-invasive imaging tools that may help to evaluate the clinical response to treatment, as well as certain new histological markers that may help in guiding the treatment planning for this atypical neoplasm.Kaposi’s sarcoma (KS) is a rare, atypical malignancy associated with immunosuppression and can be qualified as an opportunistic tumor, which responds to immune modulation or restoration. Four different epidemiological forms have been individualized (AIDS-related, iatrogenic, endemic or classic KS). Although clinical examination is sufficient to diagnose cutaneous lesions of KS, additional explorations are necessary in order to detect lesions involving other organs. New histological markers have been developed in recent years concerning the detection of HHV-8 latent or lytic proteins in the lesions, helping to confirm the diagnosis when it is clinically doubtful. More recently, the evaluation of the local immune response has also been shown to provide some guidance in choosing the appropriate therapeutic option when necessary. We also review the indication and the results of conventional radiological imaging and of non-invasive imaging tools such as 18F-fluoro-deoxy-glucose positron emission tomography, thermography and laser Doppler imaging for the diagnosis of KS and for the follow-up of therapeutic response in patients requiring systemic treatment.

Abstract 10474: The Bet Protein Inhibitor Apabetalone Rescues Diabetes Induced Impairment of Angiogenic Response

Introduction: Therapeutic modulation of blood vessel growth is a promising therapeutic strategy for the prevention of limb ischemia in diabetic (DM) patients with peripheral artery disease (PAD). Epigenetic changes, namely posttranslational histone modifications, participate in angiogenic response suggesting that chromatin-modifying drugs could be beneficial in this setting. Apabetalone (APA), a selective inhibitor of bromodomain (BRD) and extra-terminal (BET) proteins, prevents BRD4 interactions with chromatin thus modulating transcriptional programs in different organs. Hypothesis: We sought to investigate whether APA affects angiogenic response in diabetes. Methods: Primary human aortic endothelial cells (HAECs) were exposed to normal glucose (NG, 5 mM) or high glucose (HG, 20 mM) for 48 hours in presence of RVX-208 (20μM) or vehicle (DMSO). Streptozotocin-induced diabetic mice were orally treated with APA or vehicle for 5 days. Hindlimb ischemia was induced in diabetic mice and blood flow recovery analyzed at 30 minutes, 7, &amp; 14 days by laser Doppler imaging. Gastrocnemius muscle samples from patients with and without diabetes were employed to translate our experimental findings. Results: APA restored tube formation and migration in human endothelial aortic cells (HAECs) exposed to HG levels. Expression profiling of angiogenesis genes showed that APA prevents HG-induced upregulation of the anti-angiogenic molecule thrombospondin (THBS1). ChIP-seq and chromatin immunoprecipitation (ChIP) assays in HG-treated HAECs showed the enrichment of both BRD4 and active regulatory marks (H3K27ac) on THBS1 promoter, whereas BRD4 inhibition by APA prevented chromatin accessibility and THBS1 transcription. Mechanistically, we show that THBS1 inhibits angiogenesis by suppressing VEGFA signaling, while APA prevents these detrimental changes. In diabetic mice with hindlimb ischemia, epigenetic editing by APA restored THSB1/VEGFA axis thus improving limb vascularization and perfusion. Finally, epigenetic regulation of THSB1 by BRD4/H3K27ac was also reported in DM patients with PAD as compared to non-diabetic controls. Conclusion: Our findings set the stage for clinical trials testing APA in DM patients with PAD.

Abstract 12967: Deletion of G Protein-Coupled Receptor 39 Protects Angiogenesis in Hyperglycemia

Introduction: Endothelial cells (ECs) are essential to vascular homeostasis and angiogenesis. Endothelial dysfunction significantly contributes to critical limb ischemia in patients with diabetes. G protein-coupled receptor (GPR) 39 is an orphan receptor whose role in EC functions is unknown. We hypothesize that inhibition of GPR 39 protects angiogenesis under hyperglycemic conditions. Methods and Results: Healthy human aortic endothelial cells (H-HAECs) and diabetic human aortic endothelial cells (D-HAECs) were cultured in vitro . The migration (Modified Boyden Chamber assay) and tube formation capacity of H-HAECs were impaired by the transfection of adenovirus carrying human GPR39 or by a GPR39 agonist, TC-G-1008 (both n=5, p&lt;0.05). Conversely, GPR39 siRNA improved D-HAEC migration (n=4 per group, p&lt;0.05). In an angiogenesis protein profiler array, GPR39 overexpression has significantly reduced vascular endothelial growth factor (VEGF), Interleukin-8, monocyte Chemoattractant Protein-1 (MCP1), and Angiopoietin-2 in H-HAECs (n=5 per group, p&lt;0.05). Adult male GPR39 global knockout (GPR39 KO ) mice and age- and sex-matched wild-type (GPR39 WT ) litters were rendered diet-induced obese (DIO) by a 10-week high-fat and an add-on 8-week hyperglycemia by low-dose streptozotocin (STZ) injections chow as they reflect the human condition where obesity is closely linked to type 2 diabetes development. Hind limb ischemia was created by left femoral artery ligation. Laser Doppler imaging showed that GPR39 KO mice had a faster blood perfusion recovery from ischemia than GPR39 WT mice (n=5 per group, p&lt;0.05). In adult male C57BL/6 with DIO-STZ, intramuscular injection of EC-affinitive AAV-mediated shRNA against GPR39 significantly accelerated blood perfusion recovery and prevented tissue necrosis compared to empty AAV treatment (n=5 per group, p&lt;0.05). Conclusion: Our data indicated that genetic inhibition of GPR39 improved EC functions and accelerated angiogenesis from ischemia. We believe that the investigations in endothelial GPR39 may help better identify the novel therapeutic potential of targeting GPR39 in treating vascular complications in hyperglycemia.

A Thermoresponsive Chitosan/β-Glycerophosphate Hydrogel for Minimally Invasive Treatment of Critical Limb Ischaemia.

A reduction in blood supply to any limb causes ischaemia, pain and morbidity. Critical limb ischaemia is the most serious presentation of peripheral vascular disease. One in five patients with critical limb ischaemia will die within six months of diagnosis and one in three will require amputation in this time. Improving blood flow to the limb, via the administration of angiogenic agents, could relieve pain and avoid amputation. Herein, chitosan is combined with β-glycerophosphate to form a thermoresponsive formulation (chitosan/β-GP) that will flow through a syringe and needle at room temperature but will form a gel at body temperature. The chitosan/β-GP hydrogel, with or without the angiogenic molecule desferrioxamine (DFO), was injected into the mouse hind limb, following vessel ligation, to test the ability of the formulations to induce angiogenesis. The effects of the formulations were measured using laser Doppler imaging to determine limb perfusion and CD31 staining to quantify the number of blood vessels. Twenty-eight days following induction of ischaemia, the chitosan/β-GP and chitosan/β-GP + 100 µM DFO formulations had significantly (p < 0.001 and p < 0.05, respectively) improved blood flow in the ischaemic limb compared with an untreated control. Chitosan/β-GP increased vessel number by 1.7-fold in the thigh of the ischaemic limb compared with an untreated control, while chitosan/β-GP + 100 µM DFO increased vessel number 1.8-fold. Chitosan/β-GP represents a potential minimally invasive treatment for critical limb ischaemia.

MicroRNA-126 mimic administration accelerates vascular perfusion recovery and angiogenesis in a hind limb ischemia model

Abstract Background Peripheral arterial disease caused mainly by atherosclerosis portent significant morbidity, adverse prognosis and mortality, with localized treatment approaches aiming at symptom alleviation and improvement of circulation. Recently, scientific interest has been shifted towards epigenomics, with microRNAs appearing as a future therapeutic target in ischemic cardiovascular diseases due to their potential in regulating angiogenesis. Purpose We investigated the pro-angiogenic effect of miRNA-126 mimic in an in vivo model of hind limb ischemia. Methods Ten-week-old male C57Bl/6 mice (n=20) were subjected to left femoral artery ligation and were treated with microRNA-126 mimic at a dose of 5mg/kg (Group A, n=10) or 0.2ml normal saline (Group B, n=10) on days 1, 3 and 7. Laser Doppler imaging was performed to verify successful ligation on day 0 and to evaluate differences in the ischemic-to-normal (I/N) hind limb perfusion ratio on day 7 and 28. Muscle tissue expression of microRNA-126 and vascular endothelial growth factor (VEGF) was determined via PCR. Results Following microRNA-126 mimic administration in Group A subjects, we noted a qualitative and quantitative stepwise increase in I/N hind limb perfusion ratio [Day 0: 0.354 (0.276, 0.455) vs. Day 8: 0.775 (0.700, 0.844) vs. Day 28: 0.681 (0.660, 0.896), p=0.001] (Figure 1, Panels A and B). In Group B a stepwise increase of lesser magnitude was observed in I/N hind limb perfusion ratio [Day 0: 0.267 (0.164, 0.383) vs. Day 8: 0.400 (0.338, 0.418) vs. Day 28: 0.539 (0.483, 0.603), p=0.074]. Importantly, over time changes of I/N hind limb perfusion ratio were significantly higher in group A compared to group B (p for interaction=0.005) (Figure 1, Panel B). Muscle tissue expression of microRNA-126 in the ischemic hind limb of Group A was 350-fold lower compared to the ischemic hind limb of Group B (p&amp;lt;0.001) (Figure 1, Panel C). A higher expression (14.2-fold) of VEGF in the ischemic hind limb of microRNA-126-treated mice compared to that of control group was detected (p&amp;lt;0.001) (Figure 1, Panel C). A statistically significant negative correlation was noted between microRNA-126 and VEGF tissue expression levels in the ischemic limbs of both Group A and B subjects whereas no correlation between microRNA-126 and VEGF was observed in the non-ischemic hind limbs of the entire study population (Figure 1, Panel D). Conclusion MicroRNA-126 mimic delivery in the ischemic hind limb of mice can accelerate vascular perfusion recovery via angiogenesis, which is mediated by VEGF expression. Funding Acknowledgement Type of funding sources: None. Figure 1

The BET protein inhibitor apabetalone (RVX-208) restores angiogenic response in type 1 and type 2 diabetes by transcriptional regulation of thrombospondin-1

Abstract Background Peripheral artery disease (PAD) is highly prevalent in people with type 2 diabetes and associates with chronic limb ischemia and poor prognosis. Understanding the mechanisms of impaired blood vessel growth in diabetic patients is of paramount importance to develop new angiogenic therapies in this setting. Dysregulation of epigenetic mechanisms of gene transcription in vascular cells contributes to cardiovascular disease development but is currently not targeted by therapies. Apabetalone (RVX-208) – an FDA approved small molecule inhibitor of the epigenetic readers bromodomain and extra-terminal (BET) proteins – has recently shown to modulate transcriptional programs implicated in vascular inflammation and atherosclerosis. Purpose To investigate RVX-208 effects in modulating angiogenic response and post-ischemic vascularization in diabetes. Methods Primary human aortic endothelial cells (HAECs) were exposed to normal glucose (NG, 5 mM) or high glucose (HG, 20 mM) for 48 hours in presence of RVX-208 (20μM) or vehicle (DMSO). Scratch and tube formation assays were performed to investigate the impact of RVX-208 on angiogenic properties of HAECs. T1D mice (streptozotocin-induced diabetes) and T2D mice (Lepdb/db) were orally treated with apabetalone or vehicle for 5 days. Hindlimb ischemia was induced in T1D mice &amp; blood flow recovery analysed at 30 minutes, 7 and 14 days by laser Doppler imaging. Sprouting and matrigel plug assays were performed in Lepdb/db mice. Gastrocnemius muscle samples from patients with and without T2D were employed to translate our experimental findings. Results HG impaired HAECs migration and tube formation as compared to NG, whereas treatment with RVX-208 rescued HG-induced impairment of angiogenic properties. Real time PCR arrays in HG-treated HAECs showed that RVX-208 treatment prevents the dysregulation of genes implicated in endothelial migration, sprouting and inflammation, namely the anti-angiogenic molecule thrombospondin (THBS1), VEGF-A, IL-1β, IL-6, VCAM-1, and CXCL1. Of interest, both gene silencing of BET protein (BRD4) or its pharmacological inhibition by RVX-208 reduced THBS1 expression while restoring VEGFA levels in HG-treated HAECs. ChIP assays showed the enrichment of both BRD4 and the active chromatin mark H3K27Ac on THBS1 promoter. Mechanistic experiments uncovered the inhibitory role of THBS1 on VEGFA signalling, as also confirmed by STRING analysis. Treatment of T1D mice with RVX-208 improved blood flow reperfusion and vascular density at 14 days as compared to vehicle-treated animals. Moreover, RVX-208 restored endothelial sprouting in T2D-Lepdb/db mice. Of clinical relevance, THBS1 was upregulated while VEGFA expression was reduced in gastrocnemius muscle specimens from T2D patients with PAD as compared to non-diabetic controls. Conclusion In vivo targeting of BET-proteins by RVX-208 may represents a novel therapeutic approach to boost post-ischemic neovascularization in diabetes. Funding Acknowledgement Type of funding sources: Public Institution(s). Main funding source(s): University of Zurich

Pharmacological blockade of histone methyltransferase SETD7 restores angiogenic response in experimental diabetes

Abstract Background Peripheral artery disease (PAD) is highly prevalent in patients with diabetes and associates with a high rate of limb amputation and poor prognosis. Surgical and catheter-based revascularization have failed to improve outcome in diabetic patients with PAD. Hence, a need exists to develop new treatment strategies able to promote blood vessel growth in the ischemic limb of diabetic patients. Mono-methylation of histone 3 at lysine 4 (H3K4me1) - a specific epigenetic signature induced by the methyltransferase SETD7 - favours a chromatin active and open state thus enabling the gene transcription. Purpose To investigate whether SETD7-dependent epigenetic changes modulate post-ischemic vascularization in experimental diabetes. Methodology Primary human aortic endothelial cells (HAECs) were exposed to normal glucose (NG, 5 mM) or high glucose (HG, 20 mM) concentrations for 48 hours. Unbiased gene expression profiling was performed by RNA sequencing (RNA-seq) followed by Ingenuity Pathway Analysis (IPA). In vitro angiogenic assays like migration assay &amp; tube formation assay were performed. Pharmacological blockade of SETD7 was achieved by using the highly selective inhibitor called (R)-PFI-2. T1D mice (streptozotocin-induced diabetes) was orally treated with (R)-PFI-2 and with vehicle for 21 days and followed by induction of hindlimb ischemia. Blood flow recovery was analyzed at 30 minutes, 7 and 14 days by laser doppler imaging. Gastrocnemius muscle samples from patients with and without T2D were employed to translate our experimental findings. Results RNA-seq in HG-treated HAECs revealed a profound upregulation of the methyltransferase SETD7, an enzyme involved in mono-methylation of lysine 4 at histone 3 (H3K4me1). SETD7 upregulation in HG-treated HAECs was associated with an increase of H3K4-mono-methylation levels as well as with impaired endothelial cell migration and tube formation. Of interest, both gene silencing (SETD7-siRNA) and pharmacological blockade of SETD7 by (R)-PFI-2 rescued hyperglycemia-induced impairment of angiogenic properties in HAECs. RNA-seq in HG-treated HAECs with and without SETD7 depletion unveiled an array of differentially expressed genes, which were mainly involved in blood vessel growth and angiogenic response. Among dysregulated genes, Chromatin immunoprecipitation (ChIP) assays showed that SETD7 specifically mono-methylates H3K4m1 in proximity of Semaphorin-3G (SEMA3G) promoter, thus regulating its expression. Treatment of T1D mice with (R)-PFI-2 improved blood flow reperfusion at 14 days as compared to vehicle-treated animals. Finally, SETD7/SEMA3G axis was upregulated in muscle specimens from T2D patients. Conclusion Targeting SETD7 represents a novel epigenetic-based therapy to boost neovascularization in diabetic patients with PAD. Funding Acknowledgement Type of funding sources: Public Institution(s). Main funding source(s): University of Zurich

632 Analysing Laser Doppler Images - A Modified Approach to the Assessment of Burn Depth

Abstract Aim To establish a method of quantifiably determining burn depth from Laser Doppler (LD) images of burns. Method NICE advises for LD scanning to be utilised for the assessment of intermediate depth burns, where there is doubt about burn depth following experienced clinician examination. However, these scan images do not provide a quantifiable measure of burn depth. LD determines burn perfusion, with deeper burns appear blue, and superficial burns appearing red. We retrospectively studied LD images from 110 patients over the course of 2019. Using Adobe Photoshop, Histogram software, we highlighted the burns using the selection tool, and determined the proportion of the burn that was deep/poorly perfused. We then correlated these results with time till recovery. Results Burns with a poorly perfused region of larger than 20%, had a proportional increase in time till full recovery. This proportional increase was also reflected in burns with a 30% and 40% poor perfusion. Burns with less than 20% of poor perfusion were found to recover at the same rate as superficial burns. This novel method for the measurement of burn depth will allow for the examining plastic surgeon to make a decision on treatment based on concrete and quantifiable burn depth data. Conclusions We have demonstrated the initial validity of a novel method for the quantifiable measurement of burn depth. Further study is required, to establish the validity of this novel approach to the quantifiable detection of burn depth.

Thermal, Hyperspectral, and Laser Doppler Imaging: Non-Invasive Tools for Detection of The Deep Inferior Epigastric Artery Perforators-A Prospective Comparison Study.

Perforator flaps have become one of the leading procedures in microsurgical tissue transfer. Individual defects require a tailored approach to guarantee the most effective treatment. A thorough understanding of the individual vascular anatomy and the location of prominent perforators is of utmost importance and usually requires invasive angiography or at least acoustic Doppler exploration. In this study, we aimed at evaluating different non-invasive imaging modalities as possible alternatives for perforator location detection. After a cooling phase, we performed thermal, hyperspectral and Laser Doppler imaging and visually evaluated a possible detection of the perforator for a period of five minutes with an image taken every minute. We identified the most prominent perforator of the deep inferior epigastric artery by handheld acoustic Doppler in 18 patients. The detected perforator locations were then correlated. Eighteen participants were assessed with six images each per imaging method. We could show a positive match for 94.44%, 38.89%, and 0% of patients and 92.59%, 25.93%, and 0% of images for the methods respectively compared to the handheld acoustic Doppler. Sex, age, abdominal girth, and BMI showed no correlation with a possible visual detection of the perforator in the images. Therefore, thermal imaging can yield valuable supporting data in the individualized procedure planning. Future larger cohort studies are required to better assess the full potential of modern handheld thermal imaging devices.

Optimization of capsaicin-induced dermal blood flow measurement by laser Doppler imaging in cynomolgus macaque.

Capsaicin is used in several areas of non-human primate research including allodynia and dermal blood flow (DBF). The capsaicin-induced DBF increase was measured using laser Doppler imaging (LDI), but this response is known to diminish upon repeated topical applications. Refinement of the experimental procedures could improve the rigor and reproducibility of the DBF migraine model. Optimal anatomical site in cynomolgus was determined, and conditions and experimental settings for DBF measurement using LDI were established. Then, two study design trial structures were compared. Medial thigh was the preferrable site, and an ethanol-Tween 20 formulation of capsaicin was desirable. A 1-week washout for contralateral side or 2-week washout for ipsilateral side was necessary to eradicate capsaicin desensitization. With the established technicality in DBF measurements in cynomolgus macaques, the capsaicin-induced DBF model may be utilized in translational medical research in developing migraine therapeutics.

Burn depth assessment using hyperspectral imaging in a prospective single center study

The assessment of thermal burn depth remains challenging. Over the last decades, several optical systems were developed to determine burn depth. So far, only laser doppler imaging (LDI) has been shown to be reliable while others such as infrared thermography or spectrophotometric intracutaneous analysis have been less accurate. The aim of our study is to evaluate hyperspectral imaging (HSI) as a new optical device. Patients suffering thermal trauma treated in a burn unit in Germany between November 2019 and September 2020 were included. Inclusion criteria were age ≥18 years, 2nd or 3rd degree thermal burns, written informed consent and presentation within 24 h after injury. Clinical assessment and hyperspectral imaging were performed 24, 48 and 72 h after the injury. Patients in whom secondary wound closure was complete within 21 days (group A) were compared to patients in whom secondary wound closure took more than 21 days or where skin grafting was indicated (group B). Demographic data and the primary parameters generated by HSI were documented. A Mann Whitney-U test was performed to compare the groups. A p-value below 0.05 was considered to be statistically significant. The data generated using HSI were combined to create the HSI burn index (BI). Using a logistic regression and receiver operating characteristics curve (ROC) sensitivity and specificity of the BI were calculated. The trial was officially registered on DRKS (registration number: DRKS00022843). Overall, 59 patients with burn wounds were eligible for inclusion. Ten patients were excluded because of a poor data quality. Group A comprised 36 patients with a mean age of 41.5 years and a mean burnt body surface area of 2.7%. In comparison, 13 patients were allocated to group B because of the need for a skin graft (n = 10) or protracted secondary wound closure lasting more than 21 days. The mean age of these patients was 46.8 years. They had a mean affected body surface area of 4.0%. 24, 48, and 72 h after trauma the BI was 1.0 ± 0.28, 1.2 ± 0.29 and 1.55 ± 0.27 in group A and 0.78 ± 0.14, 1.05 ± 0.23 and 1.23 ± 0.27 in group B. At every time point significant differences were demonstrated between the groups. At 24 h, ROC analysis demonstrated BI threshold of 0.95 (sensitivity 0.61/specificity 1.0), on the second day of 1.17 (sensitivity 0.51/specificity 0.81) and on the third day of 1.27 (sensitivity 0.92/specificity 0.71). Changes in microcirculation within the first 72 h after thermal trauma were reflected by an increasing BI in both groups. After 72 h, the BI is able to predict the need for a skin graft with a sensitivity of 92% and a specificity of 71%.