The spike protein of SARS-CoV-2 induces endothelial inflammation through integrin α5ß1 and NF-κB signaling.

Vascular endothelial cells (ECs) form a critical interface between blood and tissues that maintains whole-body homeostasis. In COVID-19, disruption of the EC barrier results in edema, vascular inflammation, and coagulation, hallmarks of this severe disease. However, the mechanisms by which ECs are dysregulated in COVID-19 are unclear. Here, we show that the spike protein of SARS-CoV-2 alone activates the EC inflammatory phenotype in a manner dependent on integrin ⍺5ß1 signaling. Incubation of human umbilical vein ECs with whole spike protein, its receptor-binding domain, or the integrin-binding tripeptide RGD induced the nuclear translocation of NF-κB and subsequent expression of leukocyte adhesion molecules (VCAM1 and ICAM1), coagulation factors (TF and FVIII), proinflammatory cytokines (TNFα, IL-1ß, and IL-6), and ACE2, as well as the adhesion of peripheral blood leukocytes and hyperpermeability of the EC monolayer. In addition, inhibitors of integrin ⍺5ß1 activation prevented these effects. Furthermore, these vascular effects occur in vivo, as revealed by the intravenous administration of spike, which increased expression of ICAM1, VCAM1, CD45, TNFα, IL-1ß, and IL-6 in the lung, liver, kidney, and eye, and the intravitreal injection of spike, which disrupted the barrier function of retinal capillaries. We suggest that the spike protein, through its RGD motif in the receptor-binding domain, binds to integrin ⍺5ß1 in ECs to activate the NF-κB target gene expression programs responsible for vascular leakage and leukocyte adhesion. These findings uncover a new direct action of SARS-CoV-2 on EC dysfunction and introduce integrin ⍺5ß1 as a promising target for treating vascular inflammation in COVID-19.

The HGR motif is the antiangiogenic determinant of vasoinhibin: implications for a therapeutic orally active oligopeptide.

The hormone prolactin acquires antiangiogenic and antivasopermeability properties after undergoing proteolytic cleavage to vasoinhibin, an endogenous prolactin fragment of 123 or more amino acids that inhibits the action of multiple proangiogenic factors. Preclinical and clinical evidence supports the therapeutic potential of vasoinhibin against angiogenesis-related diseases including diabetic retinopathy, peripartum cardiomyopathy, rheumatoid arthritis, and cancer. However, the use of vasoinhibin in the clinic has been limited by difficulties in its production. Here, we removed this barrier to using vasoinhibin as a therapeutic agent by showing that a short linear motif of just three residues (His46-Gly47-Arg48) (HGR) is the functional determinant of vasoinhibin. The HGR motif is conserved throughout evolution, its mutation led to vasoinhibin loss of function, and oligopeptides containing this sequence inhibited angiogenesis and vasopermeability with the same potency as whole vasoinhibin. Furthermore, the oral administration of an optimized cyclic retro-inverse vasoinhibin heptapeptide containing HGR inhibited melanoma tumor growth and vascularization in mice and exhibited equal or higher antiangiogenic potency than other antiangiogenic molecules currently used as anti-cancer drugs in the clinic. Finally, by unveiling the mechanism that obscures the HGR motif in prolactin, we anticipate the development of vasoinhibin-specific antibodies to solve the on-going challenge of measuring endogenous vasoinhibin levels for diagnostic and interventional purposes, the design of vasoinhibin antagonists for managing insufficient angiogenesis, and the identification of putative therapeutic proteins containing HGR.

Sequence optimization and glycosylation of vasoinhibin: Pitfalls of recombinant production.

Vasoinhibin belongs to a family of proteins with antiangiogenic properties derived by proteolytic cleavage from the hormone prolactin (PRL). Vasoinhibin isoforms range from the first 79 to the first 159 residues of PRL. In an attempt to increase the yield of recombinant vasoinhibin and avoid incorrect intra- and inter-disulfide bond formation, the cDNA sequence comprising the first 123 amino acids of human PRL, in which cysteine 58 was or not mutated to serine, was codon-optimized. The optimized constructs achieved a 6-fold increase in mRNA expression but showed no change in protein production and reduced protein secretion when expressed in human embryo kidney (HEK293T/17) cells. Limited vasoinhibin levels associated with the activation of the unfolded protein response (UPR) and endoplasmic reticulum-associated degradation (ERAD) as revealed by the upregulation of UPR (Bip, Xbp-1, and Chop) and ERAD (Hrd1, Os9, and Sel1l) target genes. Mutation to serine introduced a new N-glycosylation site and associated with increased glycosylation and release of glycosylated vasoinhibin isoforms having reduced antiangiogenic properties. We conclude that overexpression and excessive glycosylation lead to protein degradation and reduced bioactivity, respectively, negatively affecting the production of recombinant vasoinhibin in mammalian cells.

Immunometric and functional measurement of endogenous vasoinhibin in human sera.

Introduction: Circulating levels of the antiangiogenic protein vasoinhibin, a fragment of prolactin, are of interest in vasoproliferative retinopathies, preeclampsia, and peripartum cardiomyopathy; however, it is difficult to determine the circulating levels of vasoinhibin due to the lack of quantitative assays. Methods: This study used human serum samples to assess the concentration and bioactivity of vasoinhibin using a novel enzyme-linked immunosorbent assay (ELISA) for human vasoinhibin, which employs an anti-vasoinhibin monoclonal antibody, a human umbilical vein endothelial cell (HUVEC) proliferation assay, and a chick chorioallantoic membrane (CAM) angiogenesis assay. Results: Serum samples from 17 pregnant women without (one group) and with preeclampsia and pregnancy induced hypertension (another group) demonstrated endogenous vasoinhibin concentrations in the range of 5-340 ng/ml. Immunoactive vasoinhibin levels were significantly higher in preeclampsia serum compared to healthy pregnancy serum (mean 63.09 ± 22.15 SD vs. 19.67 ± 13.34 ng/ml, p = 0.0003), as was the bioactive vasoinhibin level as determined by the HUVEC proliferation assay (56.12 ± 19.83 vs. 13.38 ± 4.88 ng/ml, p < 0.0001). There was a correlation between the concentration of vasoinhibin measured by ELISA and the HUVEC proliferation assay (Pearson r = 0.95, p < 0.0001). Healthy serum demonstrated a proangiogenic effect in the CAM assay (p < 0.05, compared to control), while serum from preeclamptic patients demonstrated an antiangiogenic effect (p < 0.05 vs. control), as did recombinant human vasoinhibin and a synthetic circular retro-inverse vasoinhibin analogue (CRIVi45-51). The antiangiogenic effects in the CAM assay and the inhibition of HUVEC proliferation were abolished by addition of the ELISA anti-vasoinhibin monoclonal antibody, but not by mouse IgG. Discussion: These results demonstrate the first quantitation of endogenous vasoinhibin in human sera and the elevation of it levels and antiangiogenic activity in sera from women with preeclampsia. The development and implementation of a quantitative assay for vasoinhibin overcomes a long-standing barrier and suggests the thorough clinical verification of vasoinhibin as a relevant biomarker.

Vasoinhibin's Apoptotic, Inflammatory, and Fibrinolytic Actions Are in a Motif Different From Its Antiangiogenic HGR Motif.

Vasoinhibin, a proteolytic fragment of the hormone prolactin, inhibits blood vessel growth (angiogenesis) and permeability, stimulates the apoptosis and inflammation of endothelial cells, and promotes fibrinolysis. The antiangiogenic and antivasopermeability properties of vasoinhibin were recently traced to the HGR motif located in residues 46 to 48 (H46-G47-R48), allowing the development of potent, orally active, HGR-containing vasoinhibin analogues for therapeutic use against angiogenesis-dependent diseases. However, whether the HGR motif is also responsible for the apoptotic, inflammatory, and fibrinolytic properties of vasoinhibin has not been addressed. Here, we report that HGR-containing analogues are devoid of these properties. Instead, the incubation of human umbilical vein endothelial cells with oligopeptides containing the sequence HNLSSEM, corresponding to residues 30 to 36 of vasoinhibin, induced apoptosis, nuclear translocation of NF-κB, expression of genes encoding leukocyte adhesion molecules (VCAM1 and ICAM1) and proinflammatory cytokines (IL1B, IL6, and TNF), and adhesion of peripheral blood leukocytes. Also, intravenous or intra-articular injection of HNLSSEM-containing oligopeptides induced the expression of Vcam1, Icam1, Il1b, Il6, and Tnf in the lung, liver, kidney, eye, and joints of mice and, like vasoinhibin, these oligopeptides promoted the lysis of plasma fibrin clots by binding to plasminogen activator inhibitor-1 (PAI-1). Moreover, the inhibition of PAI-1, urokinase plasminogen activator receptor, or NF-κB prevented the apoptotic and inflammatory actions. In conclusion, the functional properties of vasoinhibin are segregated into 2 different structural determinants. Because apoptotic, inflammatory, and fibrinolytic actions may be undesirable for antiangiogenic therapy, HGR-containing vasoinhibin analogues stand as selective and safe agents for targeting pathological angiogenesis.

New horizons in specific hormone proteolysis.

Proteolysis of protein hormones is primarily acknowledged in the context of breakdown and metabolic clearance by hepatorenal elimination. However, less explored is the specific proteolytic processing of large protein hormones, for which canonical signaling pathways were already established [e.g., prolactin (PRL)], to generate unique messengers that impact cellular functions via pathways unrelated to the receptors of their precursor molecules. Yet, the proteolysis of PRL to generate new messengers evolved under positive selection, and cleaved protein hormones regulate essential functions to maintain homeostasis at the organismal, tissue, or organ levels. The cleavage sites at which proteolysis occurs and the proteases with their determinants define a hormone-metabolism junction at which specific proteolytic cleavage, pathological alteration, and hepatorenal elimination occur.

Plasmin generates vasoinhibin-like peptides by cleaving prolactin and placental lactogen.

Vasoinhibin is an antiangiogenic, profibrinolytic peptide generated by the proteolytic cleavage of the pituitary hormone prolactin by cathepsin D, matrix metalloproteinases, and bone morphogenetic protein-1. Vasoinhibin can also be generated when placental lactogen or growth hormone are enzymatically cleaved. Here, it is investigated whether plasmin cleaves human prolactin and placental lactogen to generate vasoinhibin-like peptides. Co-incubation of prolactin and placental lactogen with plasmin was performed and analyzed by gel electrophoresis and Western blotting. Mass spectrometric analyses were carried out for sequence validation and precise cleavage site identification. The cleavage sites responsible for the generation of the vasoinhibin-like peptides were located at K170-E171 in prolactin and R160-T161 in placental lactogen. Various genetic variants of the human prolactin and placental lactogen genes are projected to affect proteolytic generation of the vasoinhibin-like peptides. The endogenous counterparts of the vasoinhibin-like peptides generated by plasmin may represent vasoinhibin-isoforms with inhibitory effects on vasculature and coagulation.

Thrombin Cleaves Prolactin Into a Potent 5.6-kDa Vasoinhibin: Implication for Tissue Repair.

Vasoinhibin is an endogenous prolactin (PRL) fragment with profibrinolytic, antivasopermeability, and antiangiogenic effects. The fact that blood clotting, vascular permeability, and angiogenesis are functionally linked during the wound-healing process led us to investigate whether thrombin, a major protease in tissue repair, generates vasoinhibin. Here, we have incubated human PRL with thrombin and analyzed the resulting proteolytic products by Western blot, mass spectrometry, high-performance liquid chromatography purification, recombinant production, and bioactivity. We unveil a main thrombin cleavage site at R48-G49 that rapidly (< 10 minutes) generates a 5.6-kDa fragment (residues 1-48) with full vasoinhibin activity, that is, it inhibited the proliferation, invasion, and permeability of cultured endothelial cells and promoted the lysis of a fibrin clot in plasma with a similar potency to that of a conventional 14-kDa vasoinhibin (residues 1-123). The R48-G49 cleavage site is highly conserved throughout evolution and precedes the intramolecular disulfide bond (C58-C174), thereby allowing the 5.6-kDa vasoinhibin to be released without a reduction step. Furthermore, the 5.6-kDa vasoinhibin is produced by endogenous thrombin during the clotting process. These findings uncover the smallest vasoinhibin known, add thrombin to the list of PRL-cleaving proteases generating vasoinhibin, and introduce vasoinhibin as a thrombin-activated mechanism for the regulation of hemostasis, vasopermeability, and angiogenesis in response to tissue injury.

Development of Vasoinhibin-Specific Monoclonal Antibodies.

Vasoinhibin is a protein hormone with antiangiogenic, antivasodilatatory, and antivasopermeability effects generated by the proteolytic cleavage of prolactin. The discovery of its role in diabetic retinopathy and peripartum cardiomyopathy led to the evaluation of new pharmacological treatments in clinical interventional trials. However, the quantitative evaluation of vasoinhibin in biological samples from patients has not been possible due to the lack of vasoinhibin-specific antibodies. Recently, loop 1 of vasoinhibin was identified to have a different three-dimensional structure compared to PRL, and thus to contain vasoinhibin-specific epitopes. Here, we report the development of two sets of vasoinhibin-specific monoclonal antibodies against two neighboring regions of the vasoinhibin loop 1. An experimental sandwich ELISA with two monoclonal anti-vasoinhibin antibodies was developed, which had no cross-reactivity to recombinant human full-length prolactin. The ELISA had a quantitation limit of 100 ng/ml, and intra-assay- and inter-assay coefficients of variation of 12.5% and 14%, respectively. The evaluation of 15 human serum samples demonstrated concentrations of below limit of detection (n=3), below limit of quantitation (n=1) and between 0.23 µg/ml (230 ng/ml) to 605 µg/ml (n=12) in the quantifiable range. Despite the high specificity of the monoclonal-monoclonal antibody sandwiches which discriminate vasoinhibin from PRL, there might be cross-reactivities by serum proteins other than vasoinhibin. A fully established vasoinhibin ELISA may support diagnostic and therapeutic measures in vascular diseases.

Levosulpiride Increases the Levels of Prolactin and Antiangiogenic Vasoinhibin in the Vitreous of Patients with Proliferative Diabetic Retinopathy.

Purpose: High circulating levels of the hormone prolactin (PRL) protect against experimental diabetic retinopathy (DR) due to the retinal accumulation of vasoinhibin, a PRL fragment that inhibits blood vessel permeability and growth. A phase 2 clinical trial is investigating a new therapy for DR based on elevating serum PRL levels with levosulpiride, a prokinetic dopamine D2 receptor blocker. Here, we tested whether levosulpiride-induced hyperprolactinemia elevates PRL and vasoinhibin in the vitreous of volunteer patients with proliferative DR (PDR) undergoing elective pars plana vitrectomy. Methods: Patients were randomized to receive placebo (lactose pill, orally TID; n = 19) or levosulpiride (25 mg orally TID; n = 18) for the 7 days before vitrectomy. Vitreous samples from untreated non-diabetic (n = 10) and PDR (n = 17) patients were also studied. Results: Levosulpiride elevated the systemic (101 ± 13 [SEM] vs. 9.2 ± 1.3 ng/mL, P < 0.0001) and vitreous (3.2 ± 0.4 vs. 1.5 ± 0.2 ng/mL, P < 0.0001) levels of PRL, and both levels were directly correlated (r = 0.58, P < 0.0002). The vitreous from non-diabetic patients or from PDR patients treated with levosulpiride, but not from placebo-treated PDR patients, inhibited the basic fibroblast growth factor (bFGF)- and vascular endothelial growth factor (VEGF)-induced proliferation of endothelial cells in culture. Vasoinhibin-neutralizing antibodies reduced the vitreous antiangiogenic effect. Matrix metalloproteases (MMPs) in the vitreous cleaved PRL to vasoinhibin, and their activity was higher in non-diabetic than in PDR patients. Conclusions: Levosulpiride increases the levels of PRL in the vitreous of PDR patients and promotes its MMP-mediated conversion to vasoinhibin, which can inhibit angiogenesis in DR. Translational Relevance: These findings support the potential therapeutic benefit of levosulpiride against vision loss in diabetes.

Alternative ligands for thyroid hormone receptors.

Thyroid hormone receptors (TRs) are ligand-dependent transcription factors that activate or repress gene transcription, resulting in the regulation of numerous physiological programs. While 3,3',5-L-triiodothyronine is the TR cognate ligand, these receptors can also be activated by various alternative ligands, including endogenous and synthetic molecules capable of inducing diverse active receptor conformations that influence thyroid hormone-dependent signaling pathways. This review mainly discusses current knowledge on 3,5-diiodo-L-thyronine and 3,5,3'-triiodothyroacetic acid, two endogenous molecules that bind to TRs and regulate gene expression; and the molecular interactions between TRs and ligands, like synthetic thyromimetics developed to target specific TR isoforms for tissue-specific regulation of thyroid-related disorders, or endocrine disruptors that have allowed the design of new analogues and revealed essential amino acids for thyroid hormone binding.

Vasoinhibin comprises a three-helix bundle and its antiangiogenic domain is located within the first 79 residues.

Vasoinhibin belongs to a family of angiogenesis inhibitors generated when the fourth α-helix (H4) of the hormone prolactin (PRL) is removed by specific proteolytic cleavage. The antiangiogenic properties are absent in uncleaved PRL, indicating that conformational changes create a new bioactive domain. However, the solution structure of vasoinhibin and the location of its bioactive domain are unknown. Molecular dynamic simulation (MD) showed that the loss of H4 exposes the hydrophobic nucleus of PRL and leads to the compression of the molecule into a three-helix bundle that buries the hydrophobic nucleus again. Compression occurs by the movement of loop 1 (L1) and its interaction with α-helix 1 (H1) generating a new L1 conformation with electrostatic and hydrophobic surfaces distinct from those of PRL, that may correspond to a bioactive domain. Consistent with this model, a recombinant protein containing the first 79 amino acids comprising H1 and L1 of human PRL inhibited the proliferation and migration of endothelial cells and upregulated the vasoinhibin target genes, IL1A and ICAM1. This bioactivity was comparable to that of a conventional vasoinhibin having the 123 residues encompassing H1, L1, Η2, L2, and Η3 of human PRL. These findings extend the vasoinhibin family to smaller proteins and provide important structural information, which will aid in antiangiogenic drug development.