Dupilumab for cancer-associated refractory pruritus.

Background: Pruritus can be an intolerable symptom in patients with cancer. Type 2 inflammation, and specifically, the cytokines IL-4, IL-13, and IL-31, play major roles in the itching process. Dupilumab is an antibody against IL-4Rα, which is a common IL-4 and IL-13 receptor subunit. Blocking IL-4 and IL-13 activity reduces the synthesis of IL-31, the "itch cytokine," and receptors for these 3 cytokines are expressed on itch nerves. Dupilumab is approved for treating moderate-to-severe atopic dermatitis, of which itching is a significant symptom. Objective: The objective of this case study was to present the initial evidence of the safety and efficacy of dupilumab as a treatment for intractable malignancy-associated pruritus in 3 patients, thereby providing a basis for further investigation in a larger cohort. Methods: As a proof of concept, we used dupilumab in our center to treat 3 patients with intractable malignancy-associated pruritus. The first patient was a 73-year-old male with a history of prostate cancer, the second patient was a 75-year-old female with cutaneous T-cell lymphoma, and the third patient was a 32-year-old male with metastatic melanoma. All 3 patients experienced debilitating itching, which started at some stage after the malignancy had been diagnosed. Moreover, none of the 3 patients showed clinical evidence of atopic dermatitis or other causes of itching (eg, uremia or liver failure), and none of the 3 patients responded to conventional treatments for pruritus. Results: Biweekly treatment with dupilumab led to an immediate improvement in itching, which subsided entirely after a few doses without any significant adverse effects. Conclusion: We propose that dupilumab is a safe and effective treatment for intractable malignancy-associated pruritus, and we are currently testing it in a large cohort.

Dark pigmentation and related low FMOD expression increase IL-3 and facilitateplasmacytoid dendritic cell maturation.

According to epidemiological research, skin autoimmune diseases are more prevalent among black Americans. We postulated that pigment-producing melanocytes may contribute to local immune regulation in the microenvironment. We examined murine epidermal melanocytes in vitro to determine the role of pigment production in immune responses mediated by dendritic cell (DC) activation. Our study revealed that darkly pigmented melanocytes produce more IL-3 and the pro-inflammatory cytokines, IL-6 and TNF-α, and consequently induce plasmacytoid DC (pDC) maturation. Additionally, we demonstrate that low pigment-associated fibromodulin (FMOD) interferes with cytokine secretion and subsequent pDC maturation.

Novel targeted inhibition of the IL-5 axis for drug reaction with eosinophilia and systemic symptoms syndrome.

Background: The drug reaction with eosinophilia and systemic symptoms (DRESS) syndrome represents a severe hypersensitivity reaction. Up-to-date treatment is based on withdrawal of medication, supportive care, and immunosuppression using high-dose corticosteroid (CS) therapy. However, evidence-based data are lacking regarding second-line therapy for steroid-resistant or steroid-dependent patients. Objectives: We hypothesize that the interleukin (IL)-5 axis plays a critical role in the pathophysiology of DRESS; hence, inhibition of this signaling pathway could offer a potential therapy for steroid-dependent and/or steroid-resistant cases, and it may offer an alternative to CS therapy in certain patients more prone to CS toxicity. Methods: Herein, we collected worldwide data on DRESS cases treated with biological agents targeting the IL-5 axis. We reviewed all cases indexed in PubMed up to October 2022 and performed a total analysis including our center experience with two additional novel cases. Results: A review of the literature yielded 14 patients with DRESS who were treated with biological agents targeting the IL-5 axis as well as our two new cases. Reported patients are characterized by a female-to-male ratio of 1:1 and a mean age of 51.8 (17-87) years. The DRESS-inducing drugs, as expected from the prospective RegiSCAR study, were mostly antibiotics (7/16), as follows: vancomycin, trimethoprim-sulfamethoxazole, ciprofloxacin, piperacillin-tazobactam, and cefepime. DRESS patients were treated with anti-IL-5 agents (mepolizumab and reslizumab) or anti-IL-5 receptor (IL-5R) biologics (benralizumab). All patients have clinically improved under anti-IL-5/IL-5R biologics. Multiple doses of mepolizumab were needed to achieve clinical resolution, whereas a single dose of benralizumab was often sufficient. Relapse was noted in one patient receiving benralizumab treatment. One patient receiving benralizumab had a fatal outcome, although mortality was probably related to massive bleeding and cardiac arrest due to coronavirus disease 2019 (COVID-19) infection. Conclusion: Current treatment guidelines for DRESS are based on case reports and expert opinion. Understanding the central role of eosinophils in DRESS pathogenicity emphasizes the need for future implementation of IL-5 axis blockade as steroid-sparing agents, potential therapy to steroid-resistant cases, and perhaps an alternative to CS treatment in certain DRESS patients more prone to CS toxicity.

Fibromodulin Ablation Exacerbates the Severity of Acute Colitis.

Introduction: Epidemiological studies have associated pigment production with protection against certain human diseases. In contrast to African Americans, European descendants are more likely to suffer from angiogenesis-dependent and inflammatory diseases, such as wet age-related macular degeneration (ARMD) and ulcerative colitis (UC), respectively. Methods: In a mouse model of dextran sulfate sodium (DSS)-induced acute colitis, the effect of fibromodulin (FMOD) depletion was examined on colitis severity. Results: In this study, albino mice that produce high levels of FMOD developed less severe acute colitis compared with mice lacking in FMOD as assessed by clinical symptoms and histopathological changes. FMOD depletion affected the expression of tight junction proteins, contributing to the destruction of the epithelial barrier. Furthermore, this study revealed a stronger inflammatory response after DSS treatment in the absence of FMOD, where FMOD depletion led to an increase in activated T cells, plasmacytoid dendritic cells (pDCs), and type I interferon (IFN) production. Discussion: These findings point to FMOD as a potential biomarker of disease severity in UC among light-skinned individuals of European descent.

Sera of Neuromyelitis Optica Patients Increase BID-Mediated Apoptosis in Astrocytes.

Neuromyelitis optica (NMO) is a rare disease usually presenting with bilateral or unilateral optic neuritis with simultaneous or sequential transverse myelitis. Autoantibodies directed against aquaporin-4 (AQP4-IgG) are found in most patients. They are believed to cross the blood−brain barrier, target astrocytes, activate complement, and eventually lead to astrocyte destruction, demyelination, and axonal damage. However, it is still not clear what the primary pathological event is. We hypothesize that the interaction of AQP4-IgG and astrocytes leads to DNA damage and apoptosis. We studied the effect of sera from seropositive NMO patients and healthy controls (HCs) on astrocytes' immune gene expression and viability. We found that sera from seropositive NMO patients led to higher expression of apoptosis-related genes, including BH3-interacting domain death agonist (BID), which is the most significant differentiating gene (p < 0.0001), and triggered more apoptosis in astrocytes compared to sera from HCs. Furthermore, NMO sera increased DNA damage and led to a higher expression of immunological genes that interact with BID (TLR4 and NOD-1). Our findings suggest that sera of seropositive NMO patients might cause astrocytic DNA damage and apoptosis. It may be one of the mechanisms implicated in the primary pathological event in NMO and provide new avenues for therapeutic intervention.

Melanocyte-secreted fibromodulin constrains skin inflammation in mice injected with lupus serum.

Skin pigmentation has been linked to the development, prevalence, and severity of several immune-mediated diseases such as SLE. Here, we asked whether fibromodulin (FMOD), which is highly expressed in skin with light complexion, can explain the known differences in the magnitude of inflammation. C57 mice with different levels of pigmentation and FMOD were injected with human lupus serum to induce skin inflammation. Histopathologic studies revealed that black C57 FMOD+/+ that produce low levels of FMOD and white C57 FMOD -/- mice develop more severe inflammation compared with white FMOD +/+ mice. This study also revealed that dark pigmentation and FMOD deletion correlates with the increased numbers of Langerhans cells. Altogether, we identify low pigmentation and FMOD are linked to low severity of inflammation and approaches to promote FMOD expression should offer clinical benefit.

RIP1/RIP3/MLKL Mediates Myocardial Function Through Necroptosis in Experimental Autoimmune Myocarditis.

Cardiomyopathy often leads to dilated cardiomyopathy (DCM) when caused by viral myocarditis. Apoptosis is long considered as the principal process of cell death in cardiomyocytes, but programmed necrosis or necroptosis is recently believed to play an important role in cardiomyocyte cell death. We investigated the role of necroptosis and its interdependency with other processes of cell death, autophagy, and apoptosis in a rat system of experimental autoimmune myocarditis (EAM). We successfully created a rat model system of EAM by injecting porcine cardiac myosin (PCM) and showed that in EAM, all three forms of cell death increase considerably, resulting in the deterioration of cardiac conditions with an increase in inflammatory infiltration in cardiomyocytes. To explore whether necroptosis occurs in EAM rats independent of autophagy, we treated EAM rats with a RIP1/RIP3/MLKL kinase-mediated necroptosis inhibitor, Necrostatin-1 (Nec-1). In Nec-1 treated rats, cell death proceeds through apoptosis but has no significant effect on autophagy. In contrast, autophagy inhibitor 3-Methyl Adenine (3-MA) increases necroptosis, implying that blockage of autophagy must be compensated through necroptosis. Caspase 8 inhibitor zVAD-fmk blocks apoptosis but increases both necroptosis and autophagy. However, all necroptosis, apoptosis, and autophagy inhibitors independently reduce inflammatory infiltration in cardiomyocytes and improve cardiac conditions. Since apoptosis or autophagy is involved in many important cellular aspects, instead of suppressing these two major cell death processes, Nec1 can be developed as a potential therapeutic target for inflammatory myocarditis.

The Prominin-1-Derived Peptide Improves Cardiac Function Following Ischemia.

Myocardial infarction (MI) remains the leading cause of death in the western world. Despite advancements in interventional revascularization technologies, many patients are not candidates for them due to comorbidities or lack of local resources. Non-invasive approaches to accelerate revascularization within ischemic tissues through angiogenesis by providing Vascular Endothelial Growth Factor (VEGF) in protein or gene form has been effective in animal models but not in humans likely due to its short half-life and systemic toxicity. Here, we tested the hypothesis that PR1P, a small VEGF binding peptide that we developed, which stabilizes and upregulates endogenous VEGF, could be used to improve outcome from MI in rodents. To test this hypothesis, we induced MI in mice and rats via left coronary artery ligation and then treated animals with every other day intraperitoneal PR1P or scrambled peptide for 14 days. Hemodynamic monitoring and echocardiography in mice and echocardiography in rats at 14 days showed PR1P significantly improved multiple functional markers of heart function, including stroke volume and cardiac output. Furthermore, molecular biology and histological analyses of tissue samples showed that systemic PR1P targeted, stabilized and upregulated endogenous VEGF within ischemic myocardium. We conclude that PR1P is a potential non-invasive candidate therapeutic for MI.

Melanocytes determine angiogenesis gene expression across human tissues.

Several angiogenesis-dependent diseases, including age-related macular degeneration and infantile hemangioma, display differential prevalence among Black, as compared to White individuals. Although socioeconomic status and genetic architecture have been suggested as explaining these differences, we have recently shown that pigment production per se might be involved. For example, we have shown that the extracellular protein fibromodulin is a pro-angiogenic factor highly secreted by melanocytes in White but not Black individuals. Still, additional pigment-dependent angiogenic factors and their molecular mechanisms remain to be identified. Understanding the contribution of pigmentation to angiogenesis in health and disease is essential for precision medicine of angiogenesis-dependent diseases with racial disparity. Toward that goal, we compared the transcriptomes of Black and White individuals in three tissues with angiogenic activity, namely artery, whole blood, and skin. We identified several differentially expressed angiogenesis pathways, including artery morphogenesis, regulation of endothelial cell chemotaxis, and cellular response to vascular endothelial growth factor stimulus. We then demonstrated that the expression of key genes in these pathways is directly modulated by the degree of pigmentation. We further identified the precise pigment production pathway controlling the expression of these genes, namely melanocortin 1 receptor (MC1R) signaling. These results demonstrate pigment-mediated regulation of angiogenesis-related pathways and their driver genes across human tissues.

A novel strategy to enhance angiogenesis in vivo using the small VEGF-binding peptide PR1P.

Therapeutic angiogenesis is an experimental frontier in vascular biology that seeks to deliver angiogenic growth factors to ischemic or injured tissues to promote targeted formation of new blood vessels as an alternative approach to surgical revascularization procedures. Vascular endothelial growth factor (VEGF) is a potent angiogenic signal protein that is locally upregulated at sites of tissue injury. However, therapies aimed at increasing VEGF levels experimentally by injecting VEGF gene or protein failed to improve outcomes in human trials in part due to its short half-life and systemic toxicity. We recently designed a novel 12-amino acid peptide (PR1P) whose sequence was derived from an extracellular VEGF-binding domain of the pro-angiogenic glycoprotein prominin-1. In this study, we characterized the molecular binding properties of this novel potential therapeutic for targeted angiogenesis and provided the foundation for its use as an angiogenic molecule that can potentiate endogenous VEGF. We showed that PR1P bound VEGF directly and enhanced VEGF binding to endothelial cells and to VEGF receptors VEGFR2 and neuropilin-1. PR1P increased angiogenesis in the murine corneal micropocket assay when combined with VEGF, but had no activity without added VEGF. In addition, PR1P also enhanced angiogenesis in murine choroidal neovascularization and wound-healing models and augmented reperfusion in a murine hind-limb ischemia model. Together our data suggest that PR1P enhanced angiogenesis by potentiating the activity of endogenous VEGF. In so doing, this novel therapy takes advantage of endogenous VEGF gradients generated in injured tissues and may improve the efficacy of and avoid systemic toxicity seen with previous VEGF therapies.

Inflammation and Lymphedema Are Exacerbated and Prolonged by Neuropilin 2 Deficiency.

The vasculature influences the progression and resolution of tissue inflammation. Capillaries express vascular endothelial growth factor (VEGF) receptors, including neuropilins (NRPs), which regulate interstitial fluid flow. NRP2, a receptor of VEGFA and semaphorin (SEMA) 3F ligands, is expressed in the vascular and lymphatic endothelia. Previous studies have demonstrated that blocking VEGF receptor 2 attenuates VEGFA-induced vascular permeability. The inhibition of NRP2 was hypothesized to decrease vascular permeability as well. Unexpectedly, massive tissue swelling and edema were observed in Nrp2-/- mice compared with wild-type littermates after delayed-type hypersensitivity reactions. Vascular permeability was twofold greater in inflamed blood vessels in Nrp2-deficient mice compared to those in Nrp2-intact littermates. The addition of exogenous SEMA3F protein inhibited vascular permeability in Balb/cJ mice, suggesting that the loss of endogenous Sema3F activity in the Nrp2-deficient mice was responsible for the enhanced vessel leakage. Functional lymphatic capillaries are necessary for draining excess fluid after inflammation; however, Nrp2-mutant mice lacked superficial lymphatic capillaries, leading to 2.5-fold greater fluid retention and severe lymphedema after inflammation. In conclusion, Nrp2 deficiency increased blood vessel permeability and decreased lymphatic vessel drainage during inflammation, highlighting the importance of the NRP2/SEMA3F pathway in the modulation of tissue swelling and resolution of postinflammatory edema.

Cellular mechanism of oral absorption of solidified polymer micelles.

Oral delivery of poorly soluble and permeable drugs represents a significant challenge in drug development. The oral delivery of drugs remains to be the ultimate route of any drugs. However, in many cases, drugs are not absorbed well in the gastrointestinal tract, or they lose their activity. Polymer micelles were recognized as an effective carrier system for drug encapsulation, and are now studied as a vehicle for oral delivery of insoluble compounds. We characterized the properties of monomethoxy polyethylene glycol-poly lactic acid (mPEG-PLA) micelles, and visualized their internalization in mouse small intestine. Using Caco-2 cells as a cellular model, we studied the kinetics of particle uptake, their transport, and the molecular mechanism of their intestinal absorption. Moreover, by inhibiting specific endocytosis pathways, pharmacologically and genetically, we found that mPEG-PLA nanoparticle endocytosis is mediated by clathrin in an energy-dependent manner, and that the low-density lipoprotein receptor is involved. FROM THE CLINICAL EDITOR: Many current drugs used are non-water soluble and indeed, the ability to deliver these drugs via the gastrointestinal tract remains the holy grail for many researchers. The authors in this paper developed monomethoxy polyethylene glycol-poly lactic acid (mPEG-PLA) micelles as a drug nanocarrier, and studied the mechanism of uptake across intestinal cells. The findings should improve our current understanding and point to the development of more nanocarriers.

Mouse Retinal Whole Mounts and Quantification of Vasculature Protocol.

Angiogenesis is the formation of new blood vessels from a pre-existing vascular bed. It is a multi-step process beginning with enzymatic degradation of the capillary basement membrane, followed by endothelial cell (EC) proliferation, migration, tube formation, assembly of a new basement membrane, and pericyte stabilization. Aberrant angiogenesis plays a major role in the pathogenesis of many diseases. The regulation of this complex process is an important therapeutic target. Success in this pursuit, however, requires the development of in vivo angiogenesis models that provide a reliable and facile platform for mechanistic studies of angiogenic regulation as well as drug development and testing (Carmeliet and Jain, 2011). Postnatal development of mouse retinal vasculature offers a unique and powerful in vivo angiogenesis model because, unlike other species, mice undergo extensive angiogenesis-dependent maturation of their retinal vessels after birth. As such, this model is also very useful for the mechanistic study of embryonic vascularization (Stahl et al., 2010; Adini et al., 2003). This protocol describes the steps involved in the whole mount processing of mouse eyes for visualization of the retinal vasculature.

Melanocyte pigmentation inversely correlates with MCP-1 production and angiogenesis-inducing potential.

The incidence of certain angiogenesis-dependent diseases is higher in Caucasians than in African Americans. Angiogenesis is amplified in wound healing and cornea models in albino C57 mice compared with black C57 mice. Moreover, mouse and human melanocytes with low pigmentation stimulate endothelial cell (EC) proliferation and migration in vitro more than melanocytes with high pigmentation. This effect is due, in part, to the secretion of an angiogenic protein called fibromodulin (FMOD) from lowly pigmented melanocytes. Herein, we expand upon the mechanism contributing to increased angiogenesis in lighter skin and report that monocyte chemotactic protein-1 (MCP-1) is secreted by nonpigmented mouse melanocytes by 5- to 10-fold more than pigmented melanocytes. MCP-1 protein stimulates EC proliferation and migration in vitro and angiogenesis in vivo. Mechanistic studies determine that FMOD is upstream of MCP-1 and promotes its secretion from both melanocytes and activated ECs via stimulation of NF-κB activity. Mice injected with FMOD-neutralizing antibodies show 2.3-fold decreased levels of circulating MCP-1. Human studies confirmed that, on average, Caucasians have 2-fold higher serum levels of MCP-1 than African Americans. Taken together, this study implicates the FMOD/MCP-1 pathway in the regulation of angiogenesis by local melanocytes and suggests that melanogenic activity may protect against aberrant angiogenic diseases.

Regulation of soluble neuropilin 1, an endogenous angiogenesis inhibitor, in liver development and regeneration.

Neuropilin-1 (NRP1) is a receptor for vascular endothelial growth factor (VEGF). A soluble isoform of Nrp1 (sNrp1) has not been described in the mouse. Our goal was to examine the expression of mouse sNrp1 during liver development and regeneration.sNrp1 was cloned from mouse liver. The expression of sNrp1 and VEGF was examined in mouse liver during post-natal development and regeneration using northern blot, western blot, in situ hybridisation, and immunohistochemical analyses. HGF/NRP1 binding was examined in vitro.A novel 588-amino acid sNrp1 isoform was found to contain the ligand binding regions of Nrp1. The adult liver expressed more sNrp1 than full-length Nrp1. In vivo, hepatocytes constitutively expressed VEGF and sNrp1 in the quiescent state. sNrp1 was highly up-regulated at P20, a time point coinciding with a plateau in liver and body weights. Following hepatectomy, endogenous levels of sNrp1 decreased during the rapid growth phase, and VEGF levels were highest just prior to and during the angiogenic phase. sNrp1 levels again rose 5-10 days post-hepatectomy, presumably to control regeneration. HGF protein bound NRP1 and binding was competed with sNRP1.We cloned a novel mouse sNrp1 isoform from liver and provide evidence that this endogenous angiogenesis inhibitor may regulate VEGF or HGF bioavailability during normal physiological growth and development as well as during liver regeneration.

Melanocyte-secreted fibromodulin promotes an angiogenic microenvironment.

Studies have established that pigmentation can provide strong, protective effects against certain human diseases. For example, angiogenesis-dependent diseases such as wet age-related macular degeneration and infantile hemangioma are more common in light-skinned individuals of mixed European descent than in African-Americans. Here we found that melanocytes from light-skinned humans and albino mice secrete high levels of fibromodulin (FMOD), which we determined to be a potent angiogenic factor. FMOD treatment stimulated angiogenesis in numerous in vivo systems, including laser-induced choroidal neovascularization, growth factor-induced corneal neovascularization, wound healing, and Matrigel plug assays. Additionally, FMOD enhanced vascular sprouting during normal retinal development. Deletion of Fmod in albino mice resulted in a marked reduction in the amount of neovascularization induced by retinal vein occlusion, corneal growth factor pellets, and Matrigel plugs. Our data implicate the melanocyte-secreted factor FMOD as a key regulator of angiogenesis and suggest an underlying mechanism for epidemiological differences between light-skinned individuals of mixed European descent and African-Americans. Furthermore, inhibition of FMOD in humans has potential as a therapeutic strategy for treating angiogenesis-dependent diseases.

RhoB controls coordination of adult angiogenesis and lymphangiogenesis following injury by regulating VEZF1-mediated transcription.

Mechanisms governing the distinct temporal dynamics that characterize post-natal angiogenesis and lymphangiogenesis elicited by cutaneous wounds and inflammation remain unclear. RhoB, a stress-induced small GTPase, modulates cellular responses to growth factors, genotoxic stress and neoplastic transformation. Here we show, using RhoB null mice, that loss of RhoB decreases pathological angiogenesis in the ischaemic retina and reduces angiogenesis in response to cutaneous wounding, but enhances lymphangiogenesis following both dermal wounding and inflammatory challenge. We link these unique and opposing roles of RhoB in blood versus lymphatic vasculatures to the RhoB-mediated differential regulation of sprouting and proliferation in primary human blood versus lymphatic endothelial cells. We demonstrate that nuclear RhoB-GTP controls expression of distinct gene sets in each endothelial lineage by regulating VEZF1-mediated transcription. Finally, we identify a small-molecule inhibitor of VEZF1-DNA interaction that recapitulates RhoB loss in ischaemic retinopathy. Our findings establish the first intra-endothelial molecular pathway governing the phased response of angiogenesis and lymphangiogenesis following injury.

The albino mutation of tyrosinase alters ocular angiogenic responsiveness.

We have observed substantial differences in angiogenic responsiveness in mice and have mapped the genetic loci responsible for these differences. We have found that the albino mutation is one of the loci responsible for such differences. Using B6.A consomic strains, we determined that chromosome 7 bears a locus that inhibits VEGF-induced corneal neovascularization. F2 crosses between B6.A consomic mice and C57BL/6J parents along with AXB and BXA recombinant inbred strains demonstrated highest linkage near the tyrosinase gene. This region was named AngVq4. Congenic animals confirmed this locus, but could not demonstrate that the classical tyrosinase albino (c) mutation was causative because of the existence of additional linked loci in the congenic region. However, in 1970, a second tyrosinase albino mutation (c-2J) arose in the C57BL/6J background at Jackson Labs. Testing this strain (C57BL/6J) demonstrated that the albino mutation is sufficient to completely explain the alteration in angiogenic response that we observed in congenic animals. Thus, we conclude that the classical tyrosinase mutation is responsible for AngVq4. In contrast to the cornea, where pigmented animals exhibit increased angiogenic responsiveness, iris neovascularization was inhibited in pigmented animals. These results may partially explain increased aggressiveness in amelanotic melanoma, as well as ethnic differences in diabetic retinopathy and macular degeneration.