The Combination of Vascular Endothelial Growth Factor A (VEGF-A) and Fibroblast Growth Factor 1 (FGF1) Modified mRNA Improves Wound Healing in Diabetic Mice: An Ex Vivo and In Vivo Investigation.

BACKGROUND: 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). METHODS: 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. RESULTS: 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. CONCLUSION: f-LNPs encapsulating VEGF-A and FGF1 mRNAs present a promising approach to improving the scarring process in DFU.

Blocking phospholamban with VHH intrabodies enhances contractility and relaxation in heart failure.

The dysregulated physical interaction between two intracellular membrane proteins, the sarco/endoplasmic reticulum Ca2+ ATPase and its reversible inhibitor phospholamban, induces heart failure by inhibiting calcium cycling. While phospholamban is a bona-fide therapeutic target, approaches to selectively inhibit this protein remain elusive. Here, we report the in vivo application of intracellular acting antibodies (intrabodies), derived from the variable domain of camelid heavy-chain antibodies, to modulate the function of phospholamban. Using a synthetic VHH phage-display library, we identify intrabodies with high affinity and specificity for different conformational states of phospholamban. Rapid phenotypic screening, via modified mRNA transfection of primary cells and tissue, efficiently identifies the intrabody with most desirable features. Adeno-associated virus mediated delivery of this intrabody results in improvement of cardiac performance in a murine heart failure model. Our strategy for generating intrabodies to investigate cardiac disease combined with modified mRNA and adeno-associated virus screening could reveal unique future therapeutic opportunities.

The Effect of Platelet-Rich Plasma on Endothelial Progenitor Cell Functionality.

Platelets mediate circulating endothelial progenitor cell (EPC) recruitment and maturation, participating in vascular repair, however the underlying mechanism(s) remain unclear. We investigated the effect of platelet-rich plasma (PRP) on the functionality of CD34+-derived late-outgrowth endothelial cells (OECs) in culture. Confluent OECs were coincubated with PRP under platelet aggregation (with adenosine diphosphate; ADP) and nonaggregation conditions, in the presence/absence of the reversible P2Y12 platelet receptor antagonist ticagrelor. Outgrowth endothelial cell activation was evaluated by determining prostacyclin (PGI2) and monocyte chemoattractant protein-1 (MCP-1) release and intercellular adhesion molecule-1 (ICAM-1) membrane expression. Similar experiments were performed using human umbilical vein endothelial cells (HUVECs). Platelet-rich plasma increased ICAM-1 expression and PGI2 and MCP-1 secretion compared with autologous platelet-poor plasma, whereas ADP-aggregated platelets in PRP did not exhibit any effect. Platelet-rich plasma pretreated with ticagrelor prior to activation with ADP increased all markers to a similar extent as PRP. Similar results were obtained using HUVECs. In conclusion, PRP induces OEC activation, a phenomenon not observed when platelets are aggregated with ADP. Platelet inhibition with ticagrelor restores the PRP capability to activate OECs. Since EPC activation is important for endothelial regeneration and angiogenesis, we suggest that agents inhibiting platelet aggregation, such as ticagrelor, may promote platelet-EPC interaction and EPC function.

Model-Based Analysis Reveals a Sustained and Dose-Dependent Acceleration of Wound Healing by VEGF-A mRNA (AZD8601).

Intradermal delivery of AZD8601, an mRNA designed to produce vascular endothelial growth factor A (VEGF-A), has previously been shown to accelerate cutaneous wound healing in a murine diabetic model. Here, we develop population pharmacokinetic and pharmacodynamic models aiming to quantify the effect of AZD8601 injections on the dynamics of wound healing. A dataset of 584 open wound area measurements from 131 mice was integrated from 3 independent studies encompassing different doses, dosing timepoints, and number of doses. Evaluation of several candidate models showed that wound healing acceleration is not likely driven directly by time-dependent VEGF-A concentration. Instead, we found that administration of AZD8601 induced a sustained acceleration of wound healing depending on the accumulated dose, with a dose producing 50% of the maximal effect of 92 µg. Simulations with this model showed that a single dose of 200 µg AZD8601 can reduce the time to reach 50% wound healing by up to 5 days.

Modified VEGF-A mRNA induces sustained multifaceted microvascular response and accelerates diabetic wound healing.

Capable of mediating efficient transfection and protein production without eliciting innate immune responses, chemically modified mRNA holds great potential to produce paracrine factors at a physiologically beneficial level, in a spatiotemporally controlled manner, and with low toxicity. Although highly promising in cardiovascular medicine and wound healing, effects of this emerging therapeutic on the microvasculature and its bioactivity in disease settings remain poorly understood. Here, we longitudinally and comprehensively characterize microvascular responses to AZD8601, a modified mRNA encoding vascular endothelial growth factor A (VEGF-A), in vivo. Using multi-parametric photoacoustic microscopy, we show that intradermal injection of AZD8601 formulated in a biocompatible vehicle results in pronounced, sustained and dose-dependent vasodilation, blood flow upregulation, and neovessel formation, in striking contrast to those induced by recombinant human VEGF-A protein, a non-translatable variant of AZD8601, and citrate/saline vehicle. Moreover, we evaluate the bioactivity of AZD8601 in a mouse model of diabetic wound healing in vivo. Using a boron nanoparticle-based tissue oxygen sensor, we show that sequential dosing of AZD8601 improves vascularization and tissue oxygenation of the wound bed, leading to accelerated re-epithelialization during the early phase of diabetic wound healing.

Neprilysin Inhibits Coagulation through Proteolytic Inactivation of Fibrinogen.

Neprilysin (NEP) is an endogenous protease that degrades a wide range of peptides including amyloid beta (Aß), the main pathological component of Alzheimer's disease (AD). We have engineered NEP as a potential therapeutic for AD but found in pre-clinical safety testing that this variant increased prothrombin time (PT) and activated partial thromboplastin time (APTT). The objective of the current study was to investigate the effect of wild type NEP and the engineered variant on coagulation and define the mechanism by which this effect is mediated. PT and APTT were measured in cynomolgus monkeys and rats dosed with a human serum albumin fusion with an engineered variant of NEP (HSA-NEPv) as well as in control plasma spiked with wild type or variant enzyme. The coagulation factor targeted by NEP was determined using in vitro prothrombinase, calibrated automated thrombogram (CAT) and fibrin formation assays as well as N-terminal sequencing of fibrinogen treated with the enzyme. We demonstrate that HSA-NEP wild type and HSA-NEPv unexpectedly impaired coagulation, increasing PT and APTT in plasma samples and abolishing fibrin formation from fibrinogen. This effect was mediated through cleavage of the N-termini of the Aα- and Bß-chains of fibrinogen thereby significantly impairing initiation of fibrin formation by thrombin. Fibrinogen has therefore been identified for the first time as a substrate for NEP wild type suggesting that the enzyme may have a role in regulating fibrin formation. Reductions in NEP levels observed in AD and cerebral amyloid angiopathy may contribute to neurovascular degeneration observed in these conditions.

Boosting the coagulation restores haemostasis in ticagrelor-treated mice.

Antiplatelet therapy is given to patients with acute coronary syndrome to reduce the risk for thrombotic events, but may increase the risk for bleeding. Ticagrelor was administered intravenously to mice. Cumulative blood loss and bleeding time were measured after cutting 5 mm of the tail, 20 min after the start of ticagrelor infusion. The tail was placed in a hemoglobin-sensitive device measuring light absorbance (abs) over time for 35 min. Activated recombinant human factor VII (rhFVIIa; NovoSeven; NovoNordisk A/S, Bagsvaerd, Denmark) 1 mg/kg (study 1); recombinant human prothrombin (rhFII, MEDI8111) 10 mg/kg (study 2); or vehicle was given intravenously once bleeding had commenced, within 90s after tail cut. Ticagrelor resulted in more than 98% inhibition of ex-vivo ADP-induced platelet aggregation. In study 1, the median blood loss in the ticagrelor, vehicle, and rhFVIIa groups were 909, 122, and 397 abs*s, respectively (P < 0.05 for both comparisons, including the ticagrelor group). Similar pattern was seen for bleeding time. The median bleeding time in the ticagrelor, vehicle, and rhFVIIa groups were 2003, 449, and 884s, respectively (P < 0.05 for both comparisons, including the ticagrelor group). In study 2, the median blood loss and bleeding time in the ticagrelor group were 362 abs*s and 1847s. The corresponding numbers for the vehicle and rhFII groups were 71 abs*s and 613s, and 178 abs*s and 701s, respectively (P < 0.05 for comparisons between ticagrelor and vehicle for both blood loss and bleeding time). In mice dosed to complete P2Y12 inhibition, boosting coagulation by administration of rhFVIIa or rhFII within 90s after bleeding initiation can partly reverse ticagrelor-enhanced bleeding.

Heparin and Protamine Titration Does Not Improve Haemostasis after Cardiac Surgery: A Prospective Randomized Study.

BACKGROUND: Bleeding complications are common in cardiac surgery. Perioperative handling of heparin and protamine may influence the haemostasis. We hypothesized that heparin and protamine dosing based on individual titration curves would improve haemostasis in comparison to standard dosing. SUBJECTS AND METHODS: Sixty patients scheduled for first time elective coronary artery bypass grafting or valve surgery were included in a prospective randomized study. The patients were randomized to heparin and protamine dosing with Hepcon HMS Plus device or to standard weight and activated clotting time (ACT) based dosing. Blood samples were collected before and 10 minutes, 2 hours and 4 hours after cardiopulmonary bypass. Primary endpoint was endogenous thrombin potential in plasma 2 hours after surgery as assessed by calibrated automated thrombography. Secondary endpoints included total heparin and protamine doses, whole blood clot formation (thromboelastometry) and post-operative bleeding volume and transfusions. Heparin effect was assessed by measuring anti-Xa activity. RESULTS: Endogenous thrombin potential and clot formation deteriorated in both groups after surgery without statistically significant intergroup difference. There were no significant differences between the groups in total heparin and protamine doses, heparin effect, or postoperative bleeding and transfusions at any time point. Significant inverse correlations between anti-Xa activity and endogenous thrombin potential were observed 10 min (r = -0.43, p = 0.001), 2 hours (r = -0.66, p<0.001) and 4 hours after surgery (r = -0.58, p<0.001). CONCLUSION: In conclusion, the results suggest that perioperative heparin and protamine dosing based on individual titration curves does not improve haemostasis after cardiac surgery. Postoperative thrombin generation capacity correlates to residual heparin effect. TRIAL REGISTRATION: www.isrctn.com ISRCTN14201041.

Sustained heparin effect contributes to reduced plasma thrombin generation capacity early after cardiac surgery.

INTRODUCTION: Thrombin is a key component in the coagulation cascade, and impaired thrombin generation has been linked to increased bleeding after surgical procedures. The aim was to evaluate postoperative thrombin generation capacity in plasma after cardiac surgery, and its potential associations to activity of individual coagulation factors and heparin. MATERIAL AND METHODS: Forty-eight coronary artery bypass grafting patients were included in a prospective observational cohort study. Thrombin generation capacity was analysed in plasma with calibrated automated thrombogram with tissue factor as activator before (baseline), and 2 h and 24 h after surgery. In addition, plasma activity of coagulation factors II, V, VII, VIII, IX, X, XI, XIII, were determined. Heparin effect was assessed by anti-Xa activity, APTT and thrombin time. RESULTS: Thrombin generation was markedly reduced 2h after surgery compared to baseline. Peak levels decreased with median 74% (interquartile range 52-90), p<0.001, and endogenous thrombin generation potential decreased with 65% (43-86), p<0.001. Postoperative changes in endogenous thrombin generation potential correlated inversely to changes in anti-Xa activity (r=-0.51, p=0.010) and to changes in thrombin time (r=-0.51, p=0.009), but there were no correlations to changes in individual coagulation factor activity. CONCLUSIONS: A marked reduction in thrombin generation potential was observed in the early postoperative phase after cardiac surgery. The decrease was independent of reductions in individual coagulation factor activity but correlated to heparin effects. The results indicate that a sustained heparin effect contributes to the postoperative reduction in thrombin generation capacity.

Plasma activity of individual coagulation factors, hemodilution and blood loss after cardiac surgery: a prospective observational study.

BACKGROUND: Hemodilution and consumption of coagulation factors during cardiopulmonary bypass has been suggested to contribute to bleeding complications after cardiac surgery. The aim was to describe the activity of individual coagulation factors after CABG in relation to hemodilution and postoperative bleeding. MATERIALS AND METHODS: Plasma concentrations of fibrinogen and plasma activity of FII, FV, FVII, FVIII, FIX, FX, FXI and FXIII adjusted for hemodilution were analysed in 57 CABG patients before, and 2h and 24h after surgery. Postoperative bleeding was registered and correlations to coagulation factor activity were calculated. RESULTS: Adjusted plasma concentration of fibrinogen (-14+/-6%), and plasma activity of FII (-9+/-6%), FV (-13+/-8%), FX (-13+/-7%) and FXIII (-9+/-14%) were reduced two hours after surgery compared to baseline (all p<0.001). FVII (+3+/-12%, p=0.34) and FXI (+1+/-19%, p=0.50) were unchanged, while FVIII (+23+/-44%, p=0.006) and FIX (+23+/-17%, p<0.001) increased. Twenty-four hours after surgery fibrinogen (+45+/-27%), FVIII (+93+/-66%) and FIX (+33+/-26%) were all increased (all p<0.001), while FVII (-37+/-14%, p<0.001), FXI (-4+/-18%, p=0.02) and FXIII (-6+/-15%, p=0.004) were decreased. Median postoperative blood loss was 380 ml/12h. There were significant inverse correlations between postoperative blood loss and fibrinogen concentration 2h after surgery (r=-0.33, p=0.019) and between postoperative blood loss and pre- and postoperative FXIII activity (r=-0.34, p=0.009 and r=-0.41, p=0.003, respectively), but not between blood loss and any of the other factors. CONCLUSIONS: There is a marked dissociation in plasma activity of individual coagulation factors after CABG. Plasma concentration of fibrinogen and factor XIII activity correlates inversely to postoperative blood loss after CABG.