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.

High Resolution Continuous Elution Electrophoresis for the Evaluation of Low Abundance Serum Proteins.

BACKGROUND: The evaluation of low abundance biomarkers in the circulating low molecular weight serum proteome is an important source of information. Techniques for sample preparation to remove high abundant proteins and to enrich the low molecular weight fraction are usually required prior to novel biomarker detection. METHODS: A continuous elution electrophoresis was used to separate the low molecular weight serum proteins from the high abundance serum proteins, such as albumin and immunoglobulins. Centrifugal concentration, SDS-PAGE, and total protein staining were performed to analyze eluted protein fractions. RESULTS: Consecutive concentrated serum protein fractions demonstrate separation at a high resolution of 1 - 2 kDa below 20 kDa. CONCLUSIONS: Continuous elution electrophoresis is an adequate method to eliminate high abundance proteins which interfere with the detection of low abundance biomarkers in the low molecular weight proteome and to enrich its proteins for subsequent detection and clinical evaluation.

A comparative phylogenetic analysis of prolactin cleavage sites for the generation of vasoinhibin in vertebrates.

Vasoinhibin is a pleiotropic protein hormone with endocrine, autocrine, and paracrine effects on blood vessel growth, permeability, and dilation, and a role in several human diseases. It is generated by proteolytic cleavage of the pituitary hormone prolactin by cathepsin D. Several isoforms with a variation in the number of amino acids and corresponding molecular mass exist. This in silico study investigated the cathepsin D cleavage sites in prolactin responsible for the generation of vasoinhibin in vertebrate species. Ninety-one prolactin protein sequences from species of the taxa primates, rodents, laurasiatheria, mammals, sauropsida, and fish were retrieved, and a multiple sequence alignment was performed. Each sequence was investigated for the presence of a vasoinhibin-generating cathepsin D cleavage site and its corresponding substrate affinity using a scoring system. Primates demonstrated the highest substrate affinity for the generation of the 15 kDa vasoinhibin isoform, and fish the highest affinity for the 16.8 kDa isoform. In both cases, this associates to the presence of leucine in the cleavage site, which is not present in species of the other taxa. In primate evolution, the presence of leucine in the cleavage site occurs with the emergence of simiiformes 42 million years ago and is conserved in higher primates across all subsequent speciation nodes. The 17.2 kDa vasoinhibin isoform has a constant substrate affinity in all taxa. The presence of leucine in vasoinhibin generating cleavage sites appears as an important feature of the molecular evolution of vasoinhibin.

Matrix Metalloproteases and Cathepsin D in Human Serum do not Cleave Prolactin to Generate Vasoinhibin.

BACKGROUND: Vasoinhibin is generated in the pituitary gland and in multiple target tissues by proteolytic cleavage of prolactin by matrix metalloproteinases and cathepsin D. A dysregulation of vasoinhibin generation appears to contribute to diabetic retinopathy and diabetic macular edema, retinopathy of prematurity, peripartum cardiomyopathy, and preeclampsia. Here, we investigate whether vasoinhibin is generated by matrix metalloproteinases and cathepsin D in human serum. METHODS: The abundance of matrix metalloproteinases 1, 2, 3, 8, 9, 10, 13, tissue inhibitors of metalloproteinases 1, 2, 4, and the activity of cathepsin D in serum samples were determined. Samples from healthy male (n = 3) and female (n = 2) subjects, pregnant subjects (n = 2), and patients with type 2 diabetes mellitus (n = 2) were investigated. The samples were incubated with recombinant prolactin at 37°C, under different pH, time, and buffer conditions. Prolactin and cleaved prolactin products were investigated by SDS-PAGE and western blotting. RESULTS: Matrix metalloproteases-1, -2, -3, -8, -9, -10, -13, TIMP-1, -2, and -4, and the activity of cathepsin D were detected in all sera. Full-length prolactin incubated with human sera, containing endogenous matrix metalloproteinases and cathepsin D, remained intact at neutral pH during a time frame from 1 to 24 hours. Partial enzymatic cleavage of prolactin resulting in the generation of a vasoinhibin-like 17 kDa peptide was observed in samples incubated at pH 3.4. Heat inactivation of the serum and the addition of an MMP inhibitor suppressed the generation of the 17 kDa peptide, indicating that its generation was MMP-mediated. CONCLUSIONS: Vasoinhibin generation by enzymatic cleavage of prolactin by matrix metalloproteases or cathepsin D does not occur in human serum at physiological pH. A limited proteolysis of prolactin, resulting in the generation of a vasoinhibin-like peptide with an apparent molecular weight of 17 kDa occurs in serum at acidic pH. The generation of vasoinhibin may require the cellular and tissue microenvironments.

Higher prolactin and vasoinhibin serum levels associated with incidence and progression of retinopathy of prematurity.

BACKGROUND: Retinopathy of prematurity (ROP) is a potentially blinding, retinal neovascular disease. Systemic prolactin accesses the retina to regulate blood vessels. Prolactin is proangiogenic and can be cleaved to antiangiogenic vasoinhibins. We investigated whether circulating prolactin and vasoinhibins associate with incidence and progression of ROP. METHODS: A prospective, longitudinal, case-control study covering postnatal weeks 1 to 9 measured serum prolactin, vasoinhibins, and vascular endothelial growth factor (VEGF) weekly in 90 premature infants diagnosed as ROP or control. RESULTS: Prolactin levels were higher in ROP than in control patients before (106.2 ± 11.3 (SEM) vs. 64.7 ± 4.9 ng/ml, postnatal week 1) and during (120.6 ± 10 vs. 84.7 ± 7.5ng/ml, postnatal week 5) ROP diagnosis. Prolactin, but not gestational age, birth weight, Apgar score, sepsis, or ventilation time, correlated with ROP. The relative risk (RR) of developing ROP increased if Prolactin (PRL) levels were higher than thresholds of 80 ng/ml (RR = 1.55, 95% CI: 1.06-2.28), 100 ng/ml (RR = 1.63, 95% CI: 1.14-2.34), or 120 ng/ml (RR = 1.95, 95% CI: 1.41-2.68). Vasoinhibin levels were 39.7% higher (95% CI: 4.5-77.5) in the circulation of ROP than in control patients at postnatal week 1 and similar thereafter, whereas VEGF serum levels were always similar. CONCLUSION: High serum prolactin and vasoinhibin levels predict and may impact ROP progression.

The role of the prolactin/vasoinhibin axis in rheumatoid arthritis: an integrative overview.

Rheumatoid arthritis (RA) is a chronic, autoimmune, inflammatory disease destroying articular cartilage and bone. The female preponderance and the influence of reproductive states in RA have long linked this disease to sexually dimorphic, reproductive hormones such as prolactin (PRL). PRL has immune-enhancing properties and increases in the circulation of some patients with RA. However, PRL also suppresses the immune system, stimulates the formation and survival of joint tissues, acquires antiangiogenic properties upon its cleavage to vasoinhibins, and protects against joint destruction and inflammation in the adjuvant-induced model of RA. This review addresses risk factors for RA linked to PRL, the effects of PRL and vasoinhibins on joint tissues, blood vessels, and immune cells, and the clinical and experimental data associating PRL with RA. This information provides important insights into the pathophysiology of RA and highlights protective actions of the PRL/vasoinhibin axis that could lead to therapeutic benefits.

Vitamin D deficiency decreases survival of bacterial meningoencephalitis in mice.

BACKGROUND: Meningoencephalitis caused by Escherichia coli is associated with high rates of mortality and risk of neurological sequelae in newborns and infants and in older or immunocompromised adults. A high prevalence of neurological disorders has been observed in geriatric populations at risk of hypovitaminosis D. METHODS: In vivo, we studied the effects of vitamin D3 on survival and the host's immune response in experimental bacterial meningoencephalitis in mice after intracerebral E. coli infection. To produce different systemic vitamin D3 concentrations, mice received a low, standard, or high dietary vitamin D3 supplementation. Bacterial titers in blood, spleen, and brain homogenates were determined. Leukocyte infiltration was assessed by histological scores, and tissue cytokine or chemokine concentrations were measured. RESULTS: Mice fed a diet with low vitamin D3 concentration died earlier than control animals after intracerebral infection. Vitamin D deficiency did not inhibit leukocyte recruitment into the subarachnoid space and did not lead to an increased density of bacteria in blood, spleen, or brain homogenates. The release of proinflammatory interleukin (IL)-6 was decreased and the release of anti-inflammatory IL-10 was increased in mice fed a diet with high vitamin D3 supplementation. CONCLUSION: Our observations suggest a detrimental role of vitamin D deficiency in bacterial central nervous system infections. Vitamin D may exert immune regulatory functions.

Principles of the prolactin/vasoinhibin axis.

The hormonal family of vasoinhibins, which derive from the anterior pituitary hormone prolactin, are known for their inhibiting effects on blood vessel growth, vasopermeability, and vasodilation. As pleiotropic hormones, vasoinhibins act in multiple target organs and tissues. The generation, secretion, and regulation of vasoinhibins are embedded into the organizational principle of an axis, which integrates the hypothalamus, the pituitary, and the target tissue microenvironment. This axis is designated as the prolactin/vasoinhibin axis. Disturbances of the prolactin/vasoinhibin axis are associated with the pathogenesis of retinal and cardiac diseases and with diseases occurring during pregnancy. New phylogenetical, physiological, and clinical implications are discussed.

High Prolactin Excretion in Patients with Diabetes Mellitus and Impaired Renal Function.

BACKGROUND: The metabolic clearance of prolactin (PRL) is partially executed by the kidney. Here, we investigate the urine excretion of PRL in patients with Diabetes Mellitus and renal impairment. METHODS: Serum and urine samples were collected from male, mestizo patients in central Mexico employing a cross-sectional study design. Ninety-eight individuals had either no diabetes and normal renal function (control), diabetes and normal renal function, or diabetes with impaired renal function. PRL was determined by a chemiluminescent immunometric assay; protein, albumin, and creatinine were evaluated using quantitative colorimetric assays. The results were analyzed using ANOVA-testing. RESULTS: Patients with Diabetes Mellitus and renal impairment had significantly higher urine PRL levels than patients with Diabetes Mellitus and normal renal function and control patients. Higher urine PRL levels were associated with lower glomerular filtration rates, higher serum creatinine, and higher urinary albumin-to-creatinine ratios (UACR). Urine PRL levels correlated positively with UACR. Serum PRL levels were similar among groups. CONCLUSIONS: Patients with Diabetes Mellitus and impaired renal function demonstrate a high urinary PRL excretion. Urinary PRL excretion in the context of proteinuria could contribute to PRL dysregulation in renal impairment.

Regulation of blood vessels by prolactin and vasoinhibins.

Prolactin (PRL) stimulates the growth of new blood vessels (angiogenesis) either directly through actions on endothelial cells or indirectly by upregulating proangiogenic factors like vascular endothelial growth factor (VEGF). Moreover, PRL acquires antiangiogenic properties after undergoing proteolytic cleavage to vasoinhibins, a family of PRL fragments (including 16 kDa PRL) with potent antiangiogenic, vasoconstrictive, and antivasopermeability effects. In view of the opposing actions of PRL and vasoinhibins, the regulation of the proteases responsible for specific PRL cleavage represents an efficient mechanism for controlling blood vessel growth and function. This review briefly describes the vascular actions of PRL and vasoinhibins, and addresses how their interplay could help drive biological effects of PRL in the context of health and disease.

C-reactive protein and the acute phase reaction in geriatric patients.

The C-reactive protein (CRP), first described as a serum component capable of precipitating the C-polysaccharide of pneumococci, is one of the most important proteins because the serum concentration rises in the acute phase reaction. The acute phase reaction is the nonspecific reaction of the body to noxious stimuli of the most varied kinds, such as infections, burns, neoplasms and tissue trauma. The CRP is synthesized in liver parenchymal cells by cytokines which are derived from stimulated leucocytes and released into the circulation. Because of its molecular structure and in synergy with the complement system, it is able to precipitate and/or lyse microorganisms, thereby rendering them harmless. Measurement of the serum CRP concentration can provide important information with respect to the diagnosis and monitoring of treatment. Due to immunosenescence in geriatric patients the synthesis of CRP appears to be limited to inflammatory stimuli; however, this phenomenon does not appear to be of major clinical relevance. Despite the introduction of new parameters of the acute phase reaction, sometimes with better performance, such as interleukin-6, procalcitonin and the soluble endotoxin receptor sCD14, measurement of CRP for diagnosis and treatment monitoring is still justified even in geriatric patients as testing is rapid, economic and nearly ubiquitously available round the clock. Biochemical markers of the acute phase reaction should always be interpreted together with the clinical picture and their specific limitations.

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.

Levosulpiride for the treatment of diabetic macular oedema: a phase 2 randomized clinical trial.

BACKGROUND/OBJECTIVE: The prokinetic levosulpiride elevates vasoinhibin levels in the vitreous of patients with proliferative diabetic retinopathy (PDR) suggesting clinical benefits due to the anti-vasopermeability and anti-angiogenic properties of vasoinhibin. We investigated the biological activity of levosulpiride in centre-involving diabetic macular oedema (DME). PATIENTS/METHODS: Prospective, randomized, double-blinded, dual-centre, phase 2 trial in patients with centre-involving DME orally treated with placebo (n = 17) or levosulpiride (n = 17) for 8 weeks or in patients with PDR undergoing elective pars plana vitrectomy and receiving placebo (n = 18) or levosulpiride (n = 18) orally for the 1 week before vitrectomy. RESULTS: Levosulpiride improved changes from baseline in best-corrected visual acuity (p ≤ 0.037), central foveal thickness (CFT, p ≤ 0.013), and mean macular volume (MMV, p ≤ 0.002) at weeks 4, 6, and 8 compared to placebo. At 8 weeks, the proportion of eyes gaining ≥5 ETDRS letters at 4 m (41% vs. 28%), losing ≥21 µm in CFT (55% vs. 28%), and dropping ≥0.06 mm3 in MMV (65% vs. 29%) was higher after levosulpiride than placebo. The overall grading of visual and structural parameters improved with levosulpiride (p = 0.029). Levosulpiride reduced VEGF (p = 0.025) and PlGF (p = 0.008) levels in the vitreous of PDR patients. No significant adverse side-effects were detected. CONCLUSIONS: Oral levosulpiride for 8 weeks improved visual and structural outcomes in patients with centre-involving DME by mechanisms that may include intraocular upregulation of vasoinhibin and downregulation of VEGF and PlGF. Larger clinical trials evaluating long-term efficacy and safety are warranted.

Prolactin promotes normal liver growth, survival, and regeneration in rodents: effects on hepatic IL-6, suppressor of cytokine signaling-3, and angiogenesis.

Prolactin (PRL) is a potent liver mitogen and proangiogenic hormone. Here, we used hyperprolactinemic rats and PRL receptor-null mice (PRLR(-/-)) to study the effect of PRL on liver growth and angiogenesis before and after partial hepatectomy (PH). Liver-to-body weight ratio (LBW), hepatocyte and sinusoidal endothelial cell (SEC) proliferation, and hepatic expression of VEGF were measured before and after PH in hyperprolactinemic rats, generated by placing two anterior pituitary glands (AP) under the kidney capsule. Also, LBW and hepatic expression of IL-6, as well as suppressor of cytokine signaling-3 (SOCS-3), were evaluated in wild-type and PRLR(-/-) mice before and after PH. Hyperprolactinemia increased the LBW, the proliferation of hepatocytes and SECs, and VEGF hepatic expression. Also, liver regeneration was increased in AP-grafted rats and was accompanied by elevated hepatocyte and SEC proliferation, and VEGF expression compared with nongrafted controls. Lowering circulating PRL levels with CB-154, an inhibitor of AP PRL secretion, prevented AP-induced stimulation of liver growth. Relative to wild-type animals, PRLR(-/-) mice had smaller livers, and soon after PH, they displayed an approximately twofold increased mortality and elevated and reduced hepatic IL-6 and SOCS-3 expression, respectively. However, liver regeneration was improved in surviving PRLR(-/-) mice. PRL stimulates normal liver growth, promotes survival, and regulates liver regeneration by mechanisms that may include hepatic downregulation of IL-6 and upregulation of SOCS-3, increased hepatocyte proliferation, and angiogenesis. PRL contributes to physiological liver growth and has potential clinical utility for ensuring survival and regulating liver mass in diseases, injuries, or surgery of the liver.

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.

Melanocortin receptors in rat liver cells: change of gene expression and intracellular localization during acute-phase response.

MCRs are known to be expressed predominantly in the brain where they mediate metabolic and anti-inflammatory functions. Leptin plays an important role in appetite and energy regulation via signaling through melanocortin receptors (MCRs) in the brain. As serum levels of MCR ligands are elevated in a clinical situation [acute-phase response (APR)] to tissue damage, where the liver is responsible for the metabolic changes, we studied hepatic gene expression of MCRs in a model of muscle tissue damage induced by turpentine oil (TO) injection in rats. A significant increase in gene expression of all five MCRs (MC4R was the highest) in liver at the RNA and protein level was detected after TO injection. A similar pattern of increase was also found in the brain. Immunohistology showed MC4R in the cytoplasm, but also in the nucleus of parenchymal and non-parenchymal liver cells, whereas MC3R-positivity was mainly cytoplasmic. A time-dependent migration of MC4R protein from the cytoplasm into the nucleus was observed during APR, in parallel with an increase in α-MSH and leptin serum levels. An increase of MC4R was detected at the protein level in wild-type mice, while such an increase was not observed in IL-6ko mice during APR. Moreover, treatment of isolated liver cells with melanocortin agonists (α-MSH and THIQ) inhibited the endotoxin-induced upregulation of the acute-phase cytokine (IL-6, IL1ß and TNF-α) gene expression in Kupffer cells and of chemokine gene expression in hepatocytes. MCRs are expressed not only in the brain, but also in liver cells and their gene expression in liver and brain tissue is upregulated during APR. Due to the presence of specific ligands in the serum, they may mediate metabolic changes and exert a protective effect on liver cells.

Prolactin and vasoinhibin are endogenous players in diabetic retinopathy revisited.

Diabetic retinopathy (DR) and diabetic macular edema (DME) are major causes for visual loss in adults. Nearly half of the world's population with diabetes has some degree of DR, and DME is a major cause of visual impairment in these patients. Severe vision loss occurs because of tractional retinal detachment due to retinal neovascularization, but the most common cause of moderate vision loss occurs in DME where excessive vascular permeability leads to the exudation and accumulation of extracellular fluid and proteins in the macula. Metabolic control stands as an effective mean for controlling retinal vascular alterations in some but not all patients with diabetes, and the search of other modifiable factors affecting the risk for diabetic microvascular complications is warranted. Prolactin (PRL) and its proteolytic fragment, vasoinhibin, have emerged as endogenous regulators of retinal blood vessels. PRL acquires antiangiogenic and anti-vasopermeability properties after undergoing proteolytic cleavage to vasoinhibin, which helps restrict the vascularization of ocular organs and, upon disruption, promotes retinal vascular alterations characteristic of DR and DME. Evidence is linking PRL (and other pituitary hormones) and vasoinhibin to DR and recent preclinical and clinical evidence supports their translation into novel therapeutic approaches.

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.

Complement Proteins C5/C5a, Cathepsin D and Prolactin in Chondrocytes: A Possible Crosstalk in the Pathogenesis of Osteoarthritis.

INTRODUCTION: Both increased activity of the complement system (CS) and the role of the pituitary hormone prolactin (PRL) are implicated in osteoarthritis (OA) pathogenesis. Besides, Cathepsin D (CatD) activity is increased in the context of OA and can exert not only proteolytic but also non-proteolytic effects on cells. For the first time, possible crosstalk between two separate humoral systems: the CS and the PRL hormone systems in chondrocytes are examined together. METHODS: Primary human articular chondrocytes (hAC) were stimulated with complement protein C5 (10 µg /mL), PRL (25 ng/mL), CatD (100 ng/mL), or anaphylatoxin C5a (25 ng/mL) for 24 h or 72 h, while unstimulated cells served as controls. In addition, co-stimulations of C5 or PRL with CatD were carried out under the same conditions. The influence of the stimulants on cell viability, cell proliferation, and metabolic activity of hAC, the chondrosarcoma cell line OUMS-27, and endothelial cells of the human umbilical cord vein (HUVEC) was investigated. Gene expression analysis of C5a receptor (C5aR1), C5, complement regulatory protein CD59, PRL, PRL receptor (PRLR), CatD, and matrix metal-loproteinases (MMP)-13 were performed using real-time PCR. Also, collagen type (Col) I, Col II, C5aR1, CD59, and PRL were detected on protein level using immunofluorescence labeling. RESULTS: The stimulation of the hAC showed no significant impairment of the cell viability. C5, C5a, and PRL induced cell growth in OUMS-27 and HUVEC, but not in chondrocytes. CatD, as well as C5, significantly reduced the gene expression of CatD, C5aR1, C5, and CD59. PRLR gene expression was likewise impaired by C5, C5a, and PRL+CatD stimulation. On the protein level, CatD, as well as C5a, decreased Col II as well as C5aR1 synthesis. CONCLUSIONS: The significant suppression of the C5 gene expression under the influence of PRL+CatD and that of CD59 via PRL+/-CatD and conversely a suppression of the PRLR gene expression via C5 alone or C5a stimulation indicates an interrelation between the two mentioned systems. In addition, CatD and C5, in contrast to PRL, directly mediate possible negative feedback of their own gene expression.