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Background and Objectives : The neutrophilic peptide, alpha-defensin, is considered an evolving risk factor intimately linked with lipid mobilization. It was previously linked to augmented liver fibrosis. Here, we assess a potential association between alpha-defensin and fatty liver. Materials and Methods : A cohort of transgenic C57BL/6JDef +/+ male mice that overexpress the human neutrophil-derived alpha-defensin in their polymorphonuclear neutrophils (PMNs) were assessed for liver steatosis and fibrosis development. Wild type (C57BL/6JDef.Wt) and transgenic (C57BL/6JDef +/+ ) mice were maintained on a standard rodent chow diet for 8.5 months. At the termination of the experiment, systemic metabolic indices and hepatic immunological cell profiling were assessed. Results : The Def +/+ transgenic mice exhibited lower body and liver weights, lower serum fasting glucose and cholesterol, and significantly lower liver fat content. These results were associated with impaired liver lymphocytes count and function (lower CD8, NK cells, and killing marker CD107a). The metabolic cage demonstrated dominant fat utilization with a comparable food intake in the Def +/+ mice. Conclusions : Chronic physiological expression of alpha-defensin induces favorable blood metabolic profile, increased systemic lipolysis, and decreased hepatic fat accumulation. Further studies are needed to characterize the defensin net liver effect.

The inflammatory response to SARS/CoV-2 (COVID-19) infection may contribute to the risk of thromboembolic complications. α-Defensins, antimicrobial peptides released from activated neutrophils, are anti-fibrinolytic and prothrombotic in vitro and in mouse models. In this prospective study of 176 patients with COVID-19 infection, we found that plasma levels of α-defensins were elevated, tracked with disease progression/mortality or resolution and with plasma levels of interleukin-6 (IL-6) and D-dimers. Immunohistochemistry revealed intense deposition of α-defensins in lung vasculature and thrombi. IL-6 stimulated the release of α-defensins from neutrophils, thereby accelerating coagulation and inhibiting fibrinolysis in human blood, imitating the coagulation pattern in COVID-19 patients. The procoagulant effect of IL-6 was inhibited by colchicine, which blocks neutrophil degranulation. These studies describe a link between inflammation and the risk of thromboembolism, and they identify a potential new approach to mitigate this risk in patients with COVID-19 and potentially in other inflammatory prothrombotic conditions., (© 2021 British Society for Haematology and John Wiley & Sons Ltd.)

Atherosclerosis, the predominant cause of death in well-resourced countries, may develop in the presence of plasma lipid levels within the normal range. Inflammation may contribute to lesion development in these individuals, but the underlying mechanisms are not well understood. Transgenic mice expressing α-def-1 released from activated neutrophils develop larger lipid and macrophage-rich lesions in the proximal aortae notwithstanding hypocholesterolemia caused by accelerated clearance of α-def-1/low-density lipoprotein (LDL) complexes from the plasma. The phenotype does not develop when the release of α-def-1 is prevented with colchicine. However, ApoE-/- mice crossed with α-def-1 mice or given exogenous α-def-1 develop smaller aortic lesions associated with reduced plasma cholesterol, suggesting a protective effect of accelerated LDL clearance. Experiments were performed to address this seeming paradox and to determine if α-def-1 might provide a means to lower cholesterol and thereby attenuate atherogenesis. We confirmed that exposing ApoE-/- mice to α-def-1 lowers total plasma cholesterol and decreases lesion size. However, lesion size was larger than in mice with total plasma cholesterol lowered to the same extent by inhibiting its adsorption or by ingesting a low-fat diet. Furthermore, α-def-1 levels correlated independently with lesion size in ApoE-/- mice. These studies show that α-def-1 has competing effects on atherogenesis. Although α-def-1 accelerates LDL clearance from plasma, it also stimulates deposition and retention of LDL in the vasculature, which may contribute to development of atherosclerosis in individuals with normal or even low plasma levels of cholesterol. Inhibiting α-def-1 may attenuate the impact of chronic inflammation on atherosclerotic vascular disease., Competing Interests: The authors have declared that no competing interests exist.

Inflammation and thrombosis are integrated, mutually reinforcing processes, but the interregulatory mechanisms are incompletely defined. Here, we examined the contribution of α-defensins (α-defs), antimicrobial proteins released from activated human neutrophils, on clot formation in vitro and in vivo. Activation of the intrinsic pathway of coagulation stimulates release of α-defs from neutrophils. α-Defs accelerate fibrin polymerization, increase fiber density and branching, incorporate into nascent fibrin clots, and impede fibrinolysis in vitro. Transgenic mice (Def ++ ) expressing human α-Def-1 developed larger, occlusive, neutrophil-rich clots after partial inferior vena cava (IVC) ligation than those that formed in wild-type (WT) mice. IVC thrombi extracted from Def ++ mice were composed of a fibrin meshwork that was denser and contained a higher proportion of tightly packed compressed polyhedral erythrocytes than those that developed in WT mice. Def ++ mice were resistant to thromboprophylaxis with heparin. Inhibiting activation of the intrinsic pathway of coagulation, bone marrow transplantation from WT mice or provision of colchicine to Def ++ mice to inhibit neutrophil degranulation decreased plasma levels of α-defs, caused a phenotypic reversion characterized by smaller thrombi comparable to those formed in WT mice, and restored responsiveness to heparin. These data identify α-defs as a potentially important and tractable link between innate immunity and thrombosis., (© 2019 by The American Society of Hematology.)

Approximately one-half of the patients who develop clinical atherosclerosis have normal or only modest elevations in plasma lipids, indicating that additional mechanisms contribute to pathogenesis. In view of increasing evidence that inflammation contributes to atherogenesis, we studied the effect of human neutrophil α-defensins on low density lipoprotein (LDL) trafficking, metabolism, vascular deposition, and atherogenesis using transgenic mice expressing human α-defensins in their polymorphonuclear leukocytes (Def(+/+)). Accelerated Def(+/+) mice developed α-defensin·LDL complexes that accelerate the clearance of LDL from the circulation accompanied by enhanced vascular deposition and retention of LDL, induction of endothelial cathepsins, increased endothelial permeability to LDL, and the development of lipid streaks in the aortic roots when fed a regular diet and at normal plasma levels of LDL. Transplantation of bone marrow from Def(+/+) to WT mice increased LDL clearance, increased vascular permeability, and increased vascular deposition of LDL, whereas transplantation of WT bone marrow to Def(+/+) mice prevented these outcomes. The same outcome was obtained by treating Def(+/+) mice with colchicine to inhibit the release of α-defensins. These studies identify a potential new link between inflammation and the development of atherosclerosis., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

Rationale: The involvement of neutrophil activation in the sentinel, potentially reversible, events in the pathogenesis of acute lung injury (ALI) is only partially understood. alpha-Defensins are the most abundant proteins secreted by activated human neutrophils, but their contribution to ALI in mouse models is hindered by their absence from murine neutrophils and the inability to study their effects in isolation in other species., Objectives: To study the role of alpha-defensins in the pathogenesis of ALI in a clinically relevant setting using mice transgenic for polymorphonuclear leukocyte expression of alpha-defensins., Methods: Transgenic mice expressing polymorphonuclear leukocyte alpha-defensins were generated. ALI was induced by acid aspiration. Pulmonary vascular permeability was studied in vivo using labeled dextran and fibrin deposition. The role of the low-density lipoprotein-related receptor (LRP) in permeability was examined., Measurements and Main Results: Acid aspiration induced neutrophil migration and release of alpha-defensins into lung parenchyma and airways. ALI was more severe in alpha-defensin-expressing mice than in wild-type mice, as determined by inspection, influx of neutrophils into the interstitial space and airways, histological evidence of epithelial injury, interstitial edema, extravascular fibrin deposition, impaired oxygenation, and reduced survival. Within 4 hours of insult, alpha-defensin-expressing mice showed greater disruption of capillary-epithelial barrier function and ALI that was attenuated by systemic or intratracheal administration of specific inhibitors of the LRP., Conclusions: alpha-Defensins mediate ALI through LRP-mediated loss of capillary-epithelial barrier function, suggesting a potential new approach to intervention.

Background: Several markers of inflammation predict the risk of thrombotic cardiovascular events in patients with atherosclerosis. However, the mechanism by which vascular inflammation promotes atherothrombotic disease is incompletely understood. Human neutrophil peptides 1-3, also known as alpha-defensins, are found in human atherosclerotic arteries, inhibit LDL metabolism and fibrinolysis and promote Lp(a) binding. We asked, therefore, if alpha-defensins are risk factors for the presence and severity of atherosclerosis., Methods: alpha-Defensin was measured in skin biopsies taken from 53 male patients (age 58.7+/-11.3 years, mean+/-S.D.) immediately prior to coronary artery catheterization. Other established risk factors were measured concurrently. The correlation between alpha-defensin deposition in the skin and the severity of the coronary artery disease (CAD) was examined., Results: A statistically significant correlation was observed between the amount of alpha-defensin in skin and the severity of CAD (R=0.40, p=0.003). Multiple regression analysis showed that skin alpha-defensin is an independent predictor for CAD severity (F=4.68, p=0.035). Logistic regression analysis confirmed that skin alpha-defensin independently predicted the likelihood for CAD (p=0.016, odds estimate 5.97, 95% CL 1.4-24.2)., Conclusions: The deposition of alpha-defensin in the skin is a strong independent predictor of CAD in men. These results suggest a link between neutrophil activation and progression of atherosclerosis and provide a novel approach to assessment of risk factors for CAD.

Angiogenesis, the growth of new blood vessels, is a complex biological process that is orchestrated by several growth factors and components of the extracellular matrix, including fibronectin (FN) and its receptor the integrin alpha5beta1. Angiogenesis is a critical part of inflammation and wound repair, but the mechanism by which vascular proliferation and migration is regulated by inflammatory cells is not completely understood. We have previously shown that human neutrophil peptides (HNPs), also known as alpha-defensins, which are secreted in high concentrations when neutrophils are activated, bind specifically to FN in the extracellular matrix and inhibit plasminogen activation. Therefore, we asked whether HNPs act as a link between inflammation and angiogenesis. Alpha5beta1-mediated endothelial cell adhesion and migration to FN, both under control conditions and under stimulation by vascular endothelial growth factor (VEGF), were inhibited specifically and in a dose-dependent manner by HNPs, whereas endothelial cell adhesion and migration to other components of the extracellular matrix, such as vitronectin, collagen, or fibrinogen/fibrin were not. Consistent with this finding, HNPs bound to and promoted the binding of fibronectin to alpha5beta1 integrin in arginine-glycine-aspartic acid (RGD)-independent manner. HNPs also completely inhibited VEGF-induced proliferation and induced apoptosis of endothelial cells in a dose-dependent manner. Moreover, HNPs inhibited capillary tube formation in three-dimensional fibrin-matrices as well as neovascularization in vivo in the chicken chorioallantoic membrane assay. Taken together, these data indicate that HNPs can regulate angiogenesis by affecting endothelial cell adhesion and migration in an FN-dependent manner as well as endothelial cell proliferation. These findings provide new insight into the role of inflammatory cells in angiogenesis and might provide a platform for developing a novel class of anti-angiogenesis drugs.

We have previously identified alpha-defensin in association with medial smooth muscle cells (SMCs) in human coronary arteries. In the present paper we report that alpha-defensin, at concentrations below those found in pathological conditions, inhibits phenylephrine (PE)-induced contraction of rat aortic rings. Addition of 1 microM alpha-defensin increased the half-maximal effective concentration (EC(50)) of PE on denuded aortic rings from 32 to 630 nM. The effect of alpha-defensin was dose dependent and saturable, with a half-maximal effect at 1 microM. alpha-Defensin binds to human umbilical vein SMCs in a specific manner. The presence of 1 microM alpha-defensin inhibited the PE-mediated Ca(++) mobilization in SMCs by more than 80%. The inhibitory effect of alpha-defensin on contraction of aortic rings and Ca(++) mobilization was completely abolished by anti-low-density lipoprotein receptor-related protein/alpha(2-)macroglobulin receptor (LRP) antibodies as well as by the antagonist receptor-associated protein (RAP). alpha-Defensin binds directly to isolated LRP in a specific and dose-dependent manner; the binding was inhibited by RAP as well as by anti-LRP antibodies. alpha-Defensin is internalized by SMCs and interacts with 2 intracellular subtypes of protein kinase C (PKC) involved in muscle contraction, alpha and beta. RAP and anti-LRP antibodies inhibited the binding and internalization of alpha-defensin by SMCs and its interaction with intracellular PKCs. These observations suggest that binding of alpha-defensin to LRP expressed in SMCs leads to its internalization; internalized alpha-defensin binds to PKC and inhibits its enzymatic activity, leading to decreased Ca(++) mobilization and SMC contraction in response to PE.