Development of a multigram synthesis of the bradykinin receptor 2 agonist FR-190997 and analogs thereof.

Using Fujisawa's B2R agonist FR-190997, we recently demonstrated for the first time that agonism at the bradykinin receptor type 2 (B2R) produces substantial antiproliferative effects. FR-190997 elicited an EC50 of 80 nM in the triple-negative breast cancer cell line MDA-MB-231, a much superior performance to that exhibited by most approved breast cancer drugs. Consequently, we initiated a program aiming primarily at synthesizing adequate quantities of FR-190997 to support further in vitro and in vivo studies toward its repurposing for various cancers and, in parallel, enable the generation of novel FR-190997 analogs for an SAR study. Prerequisite for this endeavor was to address the synthetic challenges associated with the FR-190997 scaffold, which the Fujisawa chemists had constructed in 20 steps, 13 of which required chromatographic purification. We succeeded in developing a 17-step synthesis amenable to late-stage diversification that eliminated all chromatography and enabled access to multigram quantities of FR-190997 and novel derivatives thereof, supporting further anticancer research based on B2R agonists.

Evaluation of Novel B1R/B2R Agonists Containing TRIOZAN™ Nanoparticles for Targeted Brain Delivery of Antibodies in a Mouse Model of Alzheimer Disease.

The blood-brain barrier (BBB) is a major obstacle to the development of effective therapeutics for central nervous system (CNS) disorders, including Alzheimer's disease (AD). This has been particularly true in the case of monoclonal antibody (mAbs) therapeutic candidates, due to their large size. To tackle this issue, we developed new nanoformulations, comprising bio-based Triozan polymers along with kinin B1 and B2 receptor (B1R and B2R) peptide agonist analogues, as potent BBB-permeabilizers to enhance brain delivery of a new anti-C1q mAb for AD (ANX005). The prepared B1R/B2R-TRIOZAN™ nanoparticles (NPs) displayed aqueous solubility, B1R/B2R binding capacity and uniform sizes (~130-165 nm). The relative biodistribution profiles of the mAb loaded into these NPs versus the naked mAb were assessed in vivo through two routes of administrations (intravenous (IV), intranasal (IN)) in the Tg-SwDI mouse model of AD. At 24 h post-administration, brain levels of the encapsulated mAb were significantly increased (up to 12-fold (IV) and 5-fold (IN), respectively) compared with free mAb in AD brain affected regions, entorhinal cortex and hippocampus of aged mice. Liver uptakes remained relatively low with similar values for the nanoformulations and free mAb. Our findings demonstrate the potential of B1R/B2R-TRIOZAN™ NPs for the targeted delivery of new CNS drugs, which could maximize their therapeutic effectiveness.

Potentiation of B2 receptor signaling by AltB2R, a newly identified alternative protein encoded in the human bradykinin B2 receptor gene.

Recent functional and proteomic studies in eukaryotes (www.openprot.org) predict the translation of alternative open reading frames (AltORFs) in mature G-protein-coupled receptor (GPCR) mRNAs, including that of bradykinin B2 receptor (B2R). Our main objective was to determine the implication of a newly discovered AltORF resulting protein, termed AltB2R, in the known signaling properties of B2R using complementary methodological approaches. When ectopically expressed in HeLa cells, AltB2R presented predominant punctate cytoplasmic/perinuclear distribution and apparent cointeraction with B2R at plasma and endosomal/vesicular membranes. The presence of AltB2R increases intracellular [Ca2+] and ERK1/2-MAPK activation (via phosphorylation) following B2R stimulation. Moreover, HEK293A cells expressing mutant B2R lacking concomitant expression of AltB2R displayed significantly decreased maximal responses in agonist-stimulated Gαq-Gαi2/3-protein coupling, IP3 generation, and ERK1/2-MAPK activation as compared with wild-type controls. Conversely, there was no difference in cell-surface density as well as ligand-binding properties of B2R and in efficiencies of cognate agonists at promoting B2R internalization and ß-arrestin 2 recruitment. Importantly, both AltB2R and B2R proteins were overexpressed in prostate and breast cancers, compared with their normal counterparts suggesting new associative roles of AltB2R in these diseases. Our study shows that BDKRB2 is a dual-coding gene and identifies AltB2R as a novel positive modulator of some B2R signaling pathways. More broadly, it also supports a new, unexpected alternative proteome for GPCRs, which opens new frontiers in fields of GPCR biology, diseases, and drug discovery.

Antitumor activity of cell-penetrant kinin B1 receptor antagonists in human triple-negative breast cancer cells.

High nuclear expression of G protein-coupled receptors, including kinin B1 receptors (B1R), has been observed in several human cancers, but the clinical significance of this is unknown. We put forward the hypothesis that these "nuclearized" kinin B1R contribute to tumorigenicity and can be a new target in anticancer strategies. Our initial immunostaining and ultrastructural electron microscopy analyses demonstrated high B1R expression predominantly located at internal/nuclear compartments in the MDA-MB-231 triple-negative breast cancer (TNBC) cell line as well as in clinical samples of patients with TNBC. On the basis of these findings, in the present study, we evaluated the anticancer therapeutic potential of newly identified, cell-permeable B1R antagonists in MDA-MB-231 cells (ligand-receptor binding/activity assays and LC-MS/MS analyses). We found that these compounds (SSR240612, NG67, and N2000) were more toxic to MDA-MB-231 cells in comparison with low- or non-B1R expressing MCF-10A normal human mammary epithelial cells and COS-1 cells, respectively (clonogenic, MTT proliferative/cytocidal assays, and fluorescence-activated cell-sorting (FACS)-based apoptosis analyses). By comparison, the peptide B1R antagonist R954 unable to cross cell membrane failed to produce anticancer effects. Furthermore, the putative mechanisms underlying the anticancer activities of cell-penetrant B1R antagonists were assessed by analyzing cell cycle regulation and signaling molecules related to cell survival and apoptosis (FACS and western blot). Finally, drug combination experiments showed that cell-penetrant B1R antagonists can cooperate with suboptimal doses of chemotherapeutic agents (doxorubicin and paclitaxel) to promote TNBC death. This study provides evidence on the potential value of internally acting kinin B1R antagonists in averting growth of breast cancer.

Characterization of moderate tendinopathy in ex vivo stress-deprived rat tail tendons.

BACKGROUND: Stress deprivation (SD) has frequently been used as a model to study tendinopathy. Most of these studies have investigated either short-term (early tendinopathy) or long-term SD (advanced tendinopathy), while the transient mid-term SD has been given less attention. Therefore, the main objective of this study was to characterize mid-term SD. METHODS: To this end, live, healthy rat tail tendons (RTTs) were harvested and cultured without mechanical stress and then were divided into five groups based on their culture time (fresh, 2-day SD, 4-day SD, 6-day SD, and 10-day SD). For each group, the tendons were subjected to traction testing and pathohistology, immunohistochemistry, and viability assays. RESULTS: Our results showed that 4 days of SD resulted in moderate pathological changes in RTTs. These changes included increases in the space area between fibers, cell density, and fiber tortuosity as well as a decrease in collagen density and elongation of cell nuclei. No changes in the stress at failure of tendons were observed at this time point. CONCLUSIONS: This simple ex vivo model is expected to be useful for studying the progression of tendinopathy as well as for testing potential mechanobiological or pharmacological therapy strategies to stop or reverse the progression of the pathology.

Kallikrein-kinin system as the dominant mechanism to counteract hyperactive renin-angiotensin system.

The renin-angiotensin system (RAS) generates, maintains, and makes worse hypertension and cardiovascular diseases (CVDs) through its biologically active component angiotensin II (Ang II), that causes vasoconstriction, sodium retention, and structural alterations of the heart and the arteries. A few endogenous vasodilators, kinins, natriuretic peptides, and possibly angiotensin (1-7), exert opposite actions and may provide useful therapeutic agents. As endothelial autacoids, the kinins are potent vasodilators, active natriuretics, and protectors of the endothelium. Indeed, the kallikrein-kinin system (KKS) is considered the dominant mechanism for counteracting the detrimental effects of the hyperactive RAS. The 2 systems, RAS and KKS, are controlled by the angiotensin-converting enzyme (ACE) that generates Ang II and inactivates the kinins. Inhibitors of ACE can reduce the impact of Ang II and potentiate the kinins, thus contributing to restore the cardiovascular homeostasis. In the last 20 years, ACE-inhibitors (ACE-Is) have become the drugs of first choice for the treatments of the major CVDs. ACE-Is not only reduce blood pressure, as sartans also do, but by protecting and potentiating the kinins, they can reduce morbidity and mortality and improve the quality of life for patients with CVDs. This paper provides a brief review of the literature on this topic.

Kinins and peptide receptors.

This paper is divided into two sections: the first contains the essential elements of the opening lecture presented by Pr. Regoli to the 2015 International Kinin Symposium in S. Paulo, Brazil on June 28th and the second is the celebration of Dr. Regoli's 60 years of research on vasoactive peptides. The cardiovascular homeostasis derives from a balance of two systems, the renin-angiotensin system (RAS) and the kallikrein-kinin system (KKS). The biologically active effector entity of RAS is angiotensin receptor-1 (AT-1R), and that of KKS is bradykinin B2 receptor (B2R). The first mediates vasoconstriction, the second is the most potent and efficient vasodilator. Thanks to its complex and multi-functional mechanism of action, involving nitric oxide (NO), prostacyclin and endothelial hyperpolarizing factor (EDHF). B2R is instrumental for the supply of blood, oxygen and nutrition to tissues. KKS is present on the vascular endothelium and functions as an autacoid playing major roles in cardiovascular diseases (CVDs) and diabetes. KKS exerts a paramount role in the prevention of thrombosis and atherosclerosis. Such knowledge emphasizes the already prominent value of the ACE-inhibitors (ACEIs) for the treatment of CVDs and diabetes. Indeed, the ACEIs, thanks to their double action (block of the RAS and potentiation of the KKS) are the ideal agents for a rational treatment of these diseases.

Safety and pharmacokinetics of a kinin B1 receptor peptide agonist produced with different counter-ions.

Several studies have shown the potential therapeutic utility of kinin B1 receptor (B1R) peptide agonists in neurological and ischemic cardiovascular diseases and brain cancer. Preclinical safety studies are a prerequisite for further drug development. The objectives of this study were to determine the acute toxicity and pharmacokinetics of the peptide B1R agonist, SarLys[dPhe8]desArg9-bradykinin (NG29), as trifluoroacetate (TFacetate) or acetate salt form, following intravenous injection in rats. A maximum tolerated dose (MTD) of NG29-TFacetate was established at 75 mg/kg from the results of a dose range-finding study (up to 200 mg/kg). The short-term (4-day) repeat-dose toxicity study of NG29, using its MTD value, showed that NG29-acetate exhibited minimal non-adverse clinical pathology changes in hematology, coagulation, clinical chemistry and urine parameters and severe kidney histopathological changes characterized by renal tubular degeneration. No such effects were observed with NG29-TFacetate. At the injection site, NG29-TFacetate was considered to be more locally irritating when compared to the acetate form. The extent of exposure and half-life values of NG29-TFacetate were comparable to the acetate form (AUC0-α of 10.2 mg/l*h vs. 9.9 mg/l*h; T1/2 of 2.3 h vs. 2.4 h). This study shows that in rats NG29-TFacetate exhibits a superior tolerability profile compared with the peptide acetate form.

Kinin B1 receptor mediates memory impairment in the rat hippocampus.

The bradykinin (BK) receptors B1R and B2R are involved in inflammatory responses and their activation can enhance tissue damage. The B2R is constitutively expressed and mediates the physiologic effects of BK, whereas B1R expression is induced after tissue damage. Recently, they have been involved with Alzheimer's disease, ischemic stroke and traumatic brain injury (TBI). In this study, we investigated the role of bradykinin in short and long-term memory consolidation (STM and LTM). It was observed that bilateral injection of BK (300 pmol/µl) disrupted the STM consolidation but not LTM, both evaluated by inhibitory avoidance test. The STM disruption due to BK injection was blocked by the previous injection of the B1R antagonist des-Arg10-HOE140 but not by the B2R antagonist HOE140. Additionally, the injection of the B1 agonist desArg9-BK disrupted STM and LTM consolidation at doses close to physiological concentration of the peptide (2.3 and 37.5 pmol, respectively) which could be reached during tissue injury. The presence of B1R located on glial cells around the implanted guide cannula used for peptide injection was confirmed by immunofluorescence. These data imply in a possible participation of B1R in the STM impairment observed in TBI, neuroinflammation and neurodegeneration.

Kinin receptor agonism restores hindlimb postischemic neovascularization capacity in diabetic mice.

Limb ischemia is a major complication of thromboembolic diseases. Diabetes worsens prognosis by impairing neovascularization. Genetic or pharmacological inactivation of the kallikrein-kinin system aggravates limb ischemia in nondiabetic animals, whereas angiotensin I-converting enzyme/kininase II inhibition improves outcome. The role of kinins in limb ischemia in the setting of diabetes is not documented. We assessed whether selective activation of kinin receptors by pharmacological agonists can influence neovascularization in diabetic mice with limb ischemia and have a therapeutic effect. Selective pseudopeptide kinin B1 or B2 receptor agonists resistant to peptidase action were administered by osmotic minipumps at a nonhypotensive dosage for 14 days after unilateral femoral artery ligation in mice previously rendered diabetic by streptozotocin. Comparison was made with ligatured, nonagonist-treated nondiabetic and diabetic mice. Diabetes reduced neovascularization, assessed by microangiography and histologic capillary density analysis, by roughly 40%. B1 receptor agonist or B2 receptor agonist similarly restored neovascularization in diabetic mice. Neovascularization in agonist-treated diabetic mice was indistinguishable from nondiabetic mice. Both treatments restored blood flow in the ischemic hindfoot, measured by laser-Doppler perfusion imaging. Macrophage infiltration increased 3-fold in the ischemic gastrocnemius muscle during B1 receptor agonist or B2 receptor agonist treatment, and vascular endothelial growth factor (VEGF) level increased 2-fold. Both treatments increased, by 50-100%, circulating CD45/CD11b-positive monocytes and CD34(+)/VEGFR2(+) progenitor cells. Thus, selective pharmacological activation of B1 or B2 kinin receptor overcomes the effect of diabetes on postischemic neovascularization and restores tissue perfusion through monocyte/macrophage mobilization. Kinin receptors are potential therapeutic targets in limb ischemia in diabetes.

Selective kinin receptor agonists as cardioprotective agents in myocardial ischemia and diabetes.

Cardiac ischemia is a leading cause of death, especially in diabetic patients. The diabetic ischemic heart is resistant experimentally to established cardioprotective treatments. New pharmacological approaches to cardiac protection are warranted. The kallikrein-kinin system is involved in myocardial protection in ischemia. Respective roles of B1 (B1R) and B2 (B2R) receptors remain controversial. We tested whether pharmacological activation of kinin receptors may have therapeutic effect in cardiac ischemia-reperfusion in nondiabetic (NDiab) and diabetic (Diab) mice. We assessed effect on infarct size (IS) and signaling pathways involved in myocardial protection of potent selective pharmacological agonists of B1R or B2R given at reperfusion. In NDiab mice, a B2R agonist reduced IS significantly by 47%, similarly to ramiprilat or ischemic postconditioning, via activation of phosphoinositide 3 kinase/Akt pathway leading to inhibition of glycogen synthase kinase-3ß (GSK-3ß). B1R agonist had no effect on IS. In contrast, in Diab mice, the B2R agonist, ramiprilat, or ischemic postconditioning failed to reduce IS but a B1R agonist significantly reduced IS by 44% via activation of phosphoinositide 3 kinase/Akt and extracellular signal-regulated kinase 1/2, both leading to GSK-3ß inhibition. Differential effect of B2R or B1R agonists in NDiab and Diab mice can be linked to inactivation of B2R signaling and induction of B1R in heart of Diab mice. Thus, a pharmacological B2R agonist is cardioprotective in acute ischemia in nondiabetic animals. B1R agonist overcomes resistance of diabetic heart to cardioprotective treatments. Pharmacological activation of B1R and B2R may become a treatment for diabetic and nondiabetic patients, respectively, in acute coronary syndromes.

Further pharmacological evaluation of a novel synthetic peptide bradykinin B2 receptor agonist.

We recently identified a novel human B2 receptor (B2R) agonist [Hyp(3),Thi(5),(N)Chg(7),Thi(8)]-bradykinin (NG291) with greater in vitro and in vivo potency and duration of action than natural bradykinin (BK). Here, we further examined its stability and selectivity toward B2R. The hypotensive, antithrombotic, and profibrinolytic functions of NG291 relative to BK and its analogue ([Hyp(3),Thi(5),(4-Me)Tyr(8)(ΨCH(2)NH)Arg(9)]-BK) (RMP-7) were also tested. Contraction assays using isolated mouse stomachs (containing kinin B1R, B2R, and kininase I- and II-like activities) showed that NG291 is a more potent contractant than BK and is inhibited by HOE-140 (B2R antagonist) but unaffected by R954 (B1R antagonist), whereas both decreased the potency of BK. In stomach tissues from B2R knockout mice, BK maintained its activity via B1R, whereas NG291 had no contractile effect, indicating that it was selective for B2R. Unlike BK, NG291 was not degraded by rabbit lung ACE. Comparing intravenously administered BK and NG291 revealed that NG291 exhibited more potent and prolonged hypotensive action and greater antithrombotic and profibrinolytic activities. These effects were of comparable magnitude to RMP-7 and were absent in B2R knockout mice. We concluded that NG291 is a novel biostable B2R-selective agonist that may prove suitable for investigating the (pre)clinical cardioprotective efficacy of B2R activation.

Prevention of Inflammation, Neovascularization, and Retinal Dysfunction by Kinin B1 Receptor Antagonism in a Mouse Model of Age-Related Macular Degeneration.

The kallikrein-kinin system (KKS) contributes to vascular inflammation and neovascularization in age-related macular degeneration (AMD), particularly via the kinin B1 receptor (B1R). The aim of the present study was to determine the protective effects of the topical administration of the B1R antagonist (R-954) on inflammation, neovascularization, and retinal dysfunction in a murine model of neovascular AMD. Choroidal neovascularization (CNV) was induced in C57BL6 mice using an argon laser. A treatment with ocular drops of R-954 (100 µg/15 µL, twice daily in both eyes), or vehicle, was started immediately on day 0, for 7, 14, or 21 days. CNV, invasive microglia, and B1R immunoreactive glial cells, as well as electroretinography alterations, were observed within the retina and choroid of the CNV group but not in the control group. The staining of B1R was abolished by R-954 treatment as well as the proliferation of microglia. R-954 treatment prevented the CNV development (volume: 20 ± 2 vs. 152 ± 5 × 104 µm3 in R-954 vs. saline treatment). R-954 also significantly decreased photoreceptor and bipolar cell dysfunction (a-wave amplitude: -47 ± 20 vs. -34 ± 14 µV and b-wave amplitude: 101 ± 27 vs. 64 ± 17 µV in R-954 vs. saline treatment, day 7) as well as angiogenesis tufts in the retina. These results suggest that self-administration of R-954 by eye-drop treatment could be a promising therapy in AMD to preserve retinal health and vision.

Trans-arachidonic acids generated during nitrative stress induce a thrombospondin-1-dependent microvascular degeneration.

Nitrative stress has an important role in microvascular degeneration leading to ischemia in conditions such as diabetic retinopathy and retinopathy of prematurity. Thus far, mediators of nitrative stress have been poorly characterized. We recently described that trans-arachidonic acids are major products of NO(2)(*)-mediated isomerization of arachidonic acid within the cell membrane, but their biological relevance is unknown. Here we show that trans-arachidonic acids are generated in a model of retinal microangiopathy in vivo in a NO(*)-dependent manner. They induce a selective time- and concentration-dependent apoptosis of microvascular endothelial cells in vitro, and result in retinal microvascular degeneration ex vivo and in vivo. These effects are mediated by an upregulation of the antiangiogenic factor thrombospondin-1, independently of classical arachidonic acid metabolism. Our findings provide new insight into the molecular mechanisms of nitrative stress in microvascular injury and suggest new therapeutic avenues in the management of disorders involving nitrative stress, such as ischemic retinopathies and encephalopathies.

The Succinate Receptor SUCNR1 Resides at the Endoplasmic Reticulum and Relocates to the Plasma Membrane in Hypoxic Conditions.

The GPCR SUCNR1/GPR91 exerts proangiogenesis upon stimulation with the Krebs cycle metabolite succinate. GPCR signaling depends on the surrounding environment and intracellular localization through location bias. Here, we show by microscopy and by cell fractionation that in neurons, SUCNR1 resides at the endoplasmic reticulum (ER), while being fully functional, as shown by calcium release and the induction of the expression of the proangiogenic gene for VEGFA. ER localization was found to depend upon N-glycosylation, particularly at position N8; the nonglycosylated mutant receptor localizes at the plasma membrane shuttled by RAB11. This SUCNR1 glycosylation is physiologically regulated, so that during hypoxic conditions, SUCNR1 is deglycosylated and relocates to the plasma membrane. Downstream signal transduction of SUCNR1 was found to activate the prostaglandin synthesis pathway through direct interaction with COX-2 at the ER; pharmacologic antagonism of the PGE2 EP4 receptor (localized at the nucleus) was found to prevent VEGFA expression. Concordantly, restoring the expression of SUCNR1 in the retina of SUCNR1-null mice renormalized vascularization; this effect is markedly diminished after transfection of the plasma membrane-localized SUCNR1 N8A mutant, emphasizing that ER localization of the succinate receptor is necessary for proper vascularization. These findings uncover an unprecedented physiologic process where GPCR resides at the ER for signaling function.

Bradykinin protects against brain microvascular endothelial cell death induced by pathophysiological stimuli.

The morphological and functional integrity of the microcirculation is compromised in many cardiovascular diseases such as hypertension, diabetes, stroke, and sepsis. Angiotensin converting enzyme inhibitors (ACEi), which are known to favor bradykinin (BK) bioactivity by reducing its metabolism, may have a positive impact on preventing the microvascular structural rarefaction that occurs in these diseases. Our study was designed to test the hypothesis that BK, via B2 receptors (B2R), protects the viability of the microvascular endothelium exposed to the necrotic and apoptotic cell death inducers H(2)O(2) and LPS independently of hemodynamics. Expression (RT-PCR and radioligand binding) and functional (calcium mobilization with fura-2AM, and p42/p44MAPK and Akt phosphorylation assays) experiments revealed the presence of functional B2R in pig cerebral microvascular endothelial cells (pCMVEC). In vitro results showed that the cytocidal effects of H(2)O(2) and LPS on pCMVEC were significantly decreased by a BK pretreatment (MTT and crystal violet tests, annexin-V staining/FACS analysis), which was countered by the B2R antagonist HOE 140. BK treatment coincided with enhanced expression of the cytoprotective proteins COX-2, Bcl-2, and (Cu/Zn)SOD. Ex vivo assays on rat brain explants showed that BK impeded (by approximately 40%) H(2)O(2)-induced microvascular degeneration (lectin-FITC staining). The present study proposes a novel role for BK in microvascular endothelial protection, which may be pertinent to the complex mechanism of action of ACEi explaining their long-term beneficial effects in maintaining vascular integrity.

Nitric oxide and reactive oxygen species in the nucleus revisited.

Recent work from our group showed that the nuclear envelope membranes contain several G protein-coupled receptors, including prostaglandin E2 (EP3R) and endothelin-1 (ET-1) receptors. Activation of EP3R increased endothelial nitric oxide synthase (eNOS) RNA expression in nuclei. eNOS and inducible NOS (iNOS) are reported to also be present at the nuclear level. Furthermore, reactive oxygen species (ROS) were also localized at the nuclear level. In this review, we show that stimulation with NO donor sodium nitroprusside results in an increase of intranuclear calcium that was dependent on guanylate cyclase activation, but independent of MAPK. This increase in nuclear calcium correlated with an increase in nuclear transcription of iNOS. H2O2 and ET-1 increase both cytosolic and nuclear ROS in human endocardial endothelial cells and in human aortic vascular smooth muscle cells. This increase in ROS levels by H2O2 and ET-1 was reversed by the antioxidant glutathione. In addition, our results strongly suggest that cytosolic signalization is not only transmitted to the nucleus but is also generated by the nucleus. Furthermore, we demonstrate that oxidative stress can be sensed by the nucleus. These results highly suggest that ROS formation is also generated directly by the nucleus and that free radicals may contribute to ET-1 regulation of nuclear Ca2+ homeostasis.

Novel pathogenic mechanism and therapeutic approaches to angioedema associated with C1 inhibitor deficiency.

BACKGROUND: Activation of bradykinin-mediated B2 receptor has been shown to play an important role in the onset of angioedema associated with C1 inhibitor deficiency. This finding has led to the development of novel therapeutic drugs such as the B2 receptor antagonist icatibant. However, it is unclear whether other receptors expressed on endothelial cells contribute to the release of kinins and vascular leakage in these patients. The recognition of their role may have obvious therapeutic implications. OBJECTIVE: Our aim was to investigate the involvement of B1 and gC1q receptors in in vitro and in vivo models of vascular leakage induced by plasma samples obtained from patients with C1 inhibitor deficiency. METHODS: The vascular leakage was evaluated in vitro on endothelial cells by a transwell model system and in vivo on rat mesentery microvessels by intravital microscopy. RESULTS: We observed that the attack phase plasma from C1 inhibitor-deficient patients caused a delayed fluorescein-labeled albumin leakage as opposed to the rapid effect of bradykinin, whereas remission plasma elicited a modest effect compared with control plasma. The plasma permeabilizing effect was prevented by blocking the gC1q receptor-high-molecular-weight kininogen interaction, was partially inhibited by B2 receptor or B1 receptor antagonists, and was totally prevented by the mixture of the 2 antagonists. Involvement of B1 receptor was supported by the finding that albumin leakage caused by attack phase plasma was enhanced by IL-1beta and was markedly reduced by brefeldin A. CONCLUSION: Our data suggest that both B1 receptor and gC1q receptor are involved in the vascular leakage induced by hereditary and acquired angioedema plasma.

Pharmacological Modulation of Blood-Brain Barrier Permeability by Kinin Analogs in Normal and Pathologic Conditions.

The blood-brain barrier (BBB) is a major obstacle to the development of effective diagnostics and therapeutics for brain cancers and other central nervous system diseases. Peptide agonist analogs of kinin B1 and B2 receptors, acting as BBB permeabilizers, have been utilized to overcome this barrier. The purpose of the study was to provide new insights for the potential utility of kinin analogs as brain drug delivery adjuvants. In vivo imaging studies were conducted in various animal models (primary/secondary brain cancers, late radiation-induced brain injury) to quantify BBB permeability in response to kinin agonist administrations. Results showed that kinin B1 (B1R) and B2 receptors (B2R) agonists increase the BBB penetration of chemotherapeutic doxorubicin to glioma sites, with additive effects when applied in combination. B2R agonist also enabled extravasation of high-molecular-weight fluorescent dextrans (155 kDa and 2 MDa) in brains of normal mice. Moreover, a systemic single dose of B2R agonist did not increase the incidence of metastatic brain tumors originating from circulating breast cancer cells. Lastly, B2R agonist promoted the selective delivery of co-injected diagnostic MRI agent Magnevist in irradiated brain areas, depicting increased vascular B2R expression. Altogether, our findings suggest additional evidence for using kinin analogs to facilitate specific access of drugs to the brain.