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The tegument protein pp150 of Human Cytomegalovirus (HCMV) is known to be essential for the final stages of virus maturation and mediates its functions by interacting with capsid proteins. Our laboratory has previously identified the critical regions in pp150 important for pp150-capsid interactions and designed peptides similar in sequence to these regions, with a goal to competitively inhibit capsid maturation. Treatment with a specific peptide (PepCR2 or P10) targeted to pp150 conserved region 2 led to a significant reduction in murine CMV (MCMV) growth in cell culture, paving the way for in vivo testing in a mouse model of CMV infection. However, the general pharmacokinetic parameters of peptides, including rapid degradation and limited tissue and cell membrane permeability, pose a challenge to their successful use in vivo. Therefore, we designed a biopolymer-stabilized elastin-like polypeptide (ELP) fusion construct (ELP-P10) to enhance the bioavailability of P10. Antiviral efficacy and cytotoxic effects of ELP-P10 were studied in cell culture, and pharmacokinetics, biodistribution, and antiviral efficacy were studied in a mouse model of CMV infection. ELP-P10 maintained significant antiviral activity in cell culture, and this conjugation significantly enhanced P10 bioavailability in mouse tissues. The fluorescently labeled ELP-P10 accumulated to higher levels in mouse liver and kidneys as compared to the unconjugated P10. Moreover, viral titers from vital organs of MCMV-infected mice indicated a significant reduction of virus load upon ELP-P10 treatment. Therefore, ELP-P10 has the potential to be developed into an effective antiviral against CMV infection.
Assuntos
Antivirais , Infecções por Citomegalovirus , Elastina , Muromegalovirus , Peptídeos , Fosfoproteínas , Proteínas da Matriz Viral , Animais , Camundongos , Antivirais/farmacologia , Antivirais/farmacocinética , Antivirais/química , Capsídeo/metabolismo , Capsídeo/efeitos dos fármacos , Proteínas do Capsídeo/metabolismo , Proteínas do Capsídeo/química , Citomegalovirus/efeitos dos fármacos , Infecções por Citomegalovirus/tratamento farmacológico , Infecções por Citomegalovirus/virologia , Modelos Animais de Doenças , Elastina/química , Elastina/metabolismo , Polipeptídeos Semelhantes à Elastina , Muromegalovirus/efeitos dos fármacos , Peptídeos/farmacologia , Peptídeos/química , Proteínas Recombinantes de Fusão/farmacologia , Proteínas Recombinantes de Fusão/farmacocinéticaRESUMO
There is a significant need for additional therapy to improve outcomes for newborns with acute Hypoxic-ischemic (HI) encephalopathy (HIE). New evidence suggests that insulin could be neuroprotective. This study aimed to investigate whether intranasal insulin attenuates HI-induced brain damage and neurobehavioral dysfunction in neonatal rats. Postnatal day 10 (P10), Sprague-Dawley rat pups were randomly divided into Sham + Vehicle, Sham + Insulin, HI + Vehicle, and HI + Insulin groups with equal male-to-female ratios. Pups either had HI by permanent ligation of the right common carotid artery followed by 90 min of hypoxia (8% O2) or sham surgery followed by room air exposure. Immediately after HI or Sham, pups were given fluorescence-tagged insulin (Alex-546-insulin)/vehicle, human insulin (25 µg), or vehicle in each nare under anesthesia. Shortly after administration, widespread Alex-546-insulin-binding cells were detected in the brain, primarily co-localized with neuronal nuclei-positive neurons on double-immunostaining. In the hippocampus, phospho-Akt was activated in a subset of Alex-546-insulin double-labeled cells, suggesting activation of the Akt/PI3K pathway in these neurons. Intranasal insulin (InInsulin) reduced HI-induced sensorimotor behavioral disturbances at P11. InInsulin prevented HI-induced increased Fluoro-Jade C+ degenerated neurons, cleaved caspase 3+ neurons, and volume loss in the ipsilateral brain at P11. There was no sex-specific response to HI or insulin. The findings confirm that intranasal insulin provides neuroprotection against HI brain injury in P10 rats associated with activation of intracellular cell survival signaling. If further pre-clinical research shows long-term benefits, intranasal insulin has the potential to be a promising non-invasive therapy to improve outcomes for newborns with HIE.
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Inflammatory processes are activated following ischemic stroke that lead to increased tissue damage for weeks following the ischemic insult, but there are no approved therapies that target this inflammation-induced secondary injury. Here, we report that SynB1-ELP-p50i, a novel protein inhibitor of the nuclear factor kappa B (NF-κB) inflammatory cascade bound to the drug carrier elastin-like polypeptide (ELP), decreases NF-κB induced inflammatory cytokine production in cultured macrophages, crosses the plasma membrane and accumulates in the cytoplasm of both neurons and microglia in vitro, and accumulates at the infarct site where the blood-brain barrier (BBB) is compromised following middle cerebral artery occlusion (MCAO) in rats. Additionally, SynB1-ELP-p50i treatment reduces infarct volume by 11.86% compared to saline-treated controls 24 h following MCAO. Longitudinally, SynB1-ELP-p50i treatment improves survival for 14 days following stroke with no effects of toxicity or peripheral organ dysfunction. These results show high potential for ELP-delivered biologics for therapy of ischemic stroke and other central nervous system disorders and further support targeting inflammation in ischemic stroke.
Assuntos
Isquemia Encefálica , AVC Isquêmico , Acidente Vascular Cerebral , Ratos , Animais , NF-kappa B/metabolismo , AVC Isquêmico/metabolismo , Elastina/metabolismo , Encéfalo/metabolismo , Peptídeos/farmacologia , Peptídeos/metabolismo , Acidente Vascular Cerebral/metabolismo , Isquemia Encefálica/tratamento farmacológico , Isquemia Encefálica/metabolismo , Anti-Inflamatórios/farmacologia , Anti-Inflamatórios/uso terapêutico , Anti-Inflamatórios/metabolismo , Infarto da Artéria Cerebral Média/metabolismo , Inflamação/metabolismo , Microglia/metabolismoRESUMO
Inflammatory processes are activated following ischemic strokes and lead to increased tissue damage for weeks following the ischemic insult, but there are no approved therapies that target this inflammation-induced secondary injury. Here, we report that SynB1-ELP-p50i, a novel protein inhibitor of the nuclear factor kappa B (NF-κB) inflammatory cascade bound to drug carrier elastin-like polypeptide (ELP), is able to enter both neurons and microglia, cross the blood-brain barrier, localize exclusively in the ischemic core and penumbra in Wistar-Kyoto and spontaneously hypertensive rats (SHRs), and reduce infarct volume in male SHRs. Additionally, in male SHRs, SynB1-ELP-p50i treatment improves survival for 14 days following stroke with no effects of toxicity or peripheral organ dysfunction. These results show high potential for ELP-delivered biologics for therapy of ischemic stroke and other central nervous system disorders and further support targeting inflammation in ischemic stroke.
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Placenta ischemia, the initiating event in preeclampsia (PE), is associated with fetal growth restriction. Inhibition of the agonistic autoantibody against the angiotensin type 1 receptor AT1-AA, using an epitope-binding inhibitory peptide ('n7AAc') attenuates increased blood pressure at gestational day (G)19 in the clinically relevant reduced uterine perfusion pressure (RUPP) model of PE. Thus we tested the hypothesis that maternal administration of 'n7AAc' does not transfer to the fetus, improves uterine blood flow and fetal growth, and attenuates elevated placental expression of miRNAs implicated in PE and FGR. Sham or RUPP surgery was performed at G14 with vehicle or 'n7AAc' (144 µg/day) administered via an osmotic pump from G14 to G20. Maternal plasma levels of the peptide on G20 were 16.28 ± 4.4 nM, and fetal plasma levels were significantly lower at 1.15 ± 1.7 nM (P = 0.0007). The uterine artery resistance index was significantly elevated in RUPP (P < 0.0001) but was not increased in 'n7AAc'-RUPP or 'n7AAc'-Sham versus Sham. A significant reduction in fetal weight at G20 in RUPP (P = 0.003) was not observed in 'n7AAc'-RUPP. Yet, percent survival was reduced in RUPP (P = 0.0007) and 'n7AAc'-RUPP (P < 0.0002). Correlation analysis indicated the reduction in percent survival during gestation was specific to the RUPP (r = 0.5342, P = 0.043) and independent of 'n7AAc'. Placental miR-155 (P = 0.0091) and miR-181a (P = 0.0384) expression was upregulated in RUPP at G20 but was not elevated in 'n7AAc'-RUPP. Collectively, our results suggest that maternal administration of 'n7AAc' does not alter fetal growth in the RUPP implicating its potential as a therapeutic for the treatment of PE.NEW & NOTEWORTHY The seven amino acid inhibitory peptide to the AT1-AA ('n7AAc') has limited transfer to the fetus at gestational day 20, improves uterine blood flow and fetal growth in the reduced uterine perfusion pressure model of preeclampsia (PE), and does not impair fetal survival during gestation in sham-operated or placental ischemic rats. Collectively, these findings suggest that maternal administration of 'n7AAc' as an effective strategy for the treatment of PE is associated with improved outcomes in the fetus.
Assuntos
MicroRNAs , Pré-Eclâmpsia , Animais , Feminino , Humanos , Gravidez , Ratos , Aminoácidos/metabolismo , Autoanticorpos/metabolismo , Pressão Sanguínea/fisiologia , Modelos Animais de Doenças , Epitopos/metabolismo , Desenvolvimento Fetal , Isquemia , MicroRNAs/metabolismo , Peptídeos/farmacologia , Placenta/metabolismo , Ratos Sprague-Dawley , Receptor Tipo 1 de Angiotensina/metabolismo , Artéria UterinaRESUMO
The burden of acute and chronic kidney diseases to the health care system is exacerbated by the high mortality that this disease carries paired with the still limited availability of comprehensive therapies. A reason partially resides in the complexity of the kidney, with multiple potential target cell types and a complex structural environment that complicate strategies to protect and recover renal function after injury. Management of both acute and chronic renal disease, irrespective of the cause, are mainly focused on supportive treatments and renal replacement strategies when needed. Emerging preclinical evidence supports the feasibility of drug delivery technology for the kidney, and recent studies have contributed to building a robust catalog of peptides, proteins, nanoparticles, liposomes, extracellular vesicles, and other carriers that may be fused to therapeutic peptides, proteins, nucleic acids, or small molecule drugs. These fusions can display a precise renal uptake, an enhanced circulating time, and a directed intraorgan biodistribution while protecting their cargo to improve therapeutic efficacy. However, several hurdles that slow the transition towards clinical applications are still in the way, such as solubility, toxicity, and sub-optimal renal targeting. This review will discuss the feasibility and current limitations of drug delivery technologies for the treatment of renal disease, offering an update on their potential and the future directions of these promising strategies.
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Nefropatias , Nanopartículas , Sistemas de Liberação de Medicamentos , Humanos , Nefropatias/tratamento farmacológico , Lipossomos/química , Tecnologia , Distribuição TecidualRESUMO
PURPOSE: Intranasally administered unfractionated heparin (UFH) and other sulfated polysaccharides are potential prophylactics for COVID-19. The purpose of this research was to measure the safety and pharmacokinetics of clearance of intranasally administered UFH solution from the nasal cavity. METHODS: Double-blinded daily intranasal dosing in C57Bl6 mice with four doses (60 ng to 60 µg) of UFH was carried out for fourteen consecutive days, with both blood coagulation measurements and subject adverse event monitoring. The pharmacokinetics of fluorescent-labeled UFH clearance from the nasal cavity were measured in mice by in vivo imaging. Intranasal UFH at 2000 U/day solution with nasal spray device was tested for safety in a small number of healthy human subjects. RESULTS: UFH showed no evidence of toxicity in mice at any dose measured. No significant changes were observed in activated partial thromboplastin time (aPTT), platelet count, or frequency of minor irritant events over vehicle-only control. Human subjects showed no significant changes in aPTT time, international normalized ratio (INR), or platelet count over baseline measurements. No serious adverse events were observed. In vivo imaging in a mouse model showed a single phase clearance of UFH from the nasal cavity. After 12 h, 3.2% of the administered UFH remained in the nasal cavity, decaying to background levels by 48 h. CONCLUSIONS: UFH showed no toxic effects for extended daily intranasal dosing in mice as well as humans. The clearance kinetics of intranasal heparin solution from the nasal cavity indicates potentially protective levels for up to 12 h after dosing.
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COVID-19 , Heparina , Animais , Anticoagulantes/efeitos adversos , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Tempo de Tromboplastina ParcialRESUMO
PURPOSE: Intranasally administered unfractionated heparin (UFH) and other sulfated polysaccharides are potential prophylactics for COVID-19. The purpose of this research was to measure the safety and pharmacokinetics of clearance of intranasally administered UFH solution from the nasal cavity. METHODS: Double-blinded daily intranasal dosing in C57Bl6 mice with four doses (60 ng to 60 µg) of UFH was carried out for fourteen consecutive days, with both blood coagulation measurements and subject adverse event monitoring. The pharmacokinetics of fluorescent-labeled UFH clearance from the nasal cavity were measured in mice by in vivo imaging. Intranasal UFH at 2000 U/day solution with nasal spray device was tested for safety in a small number of healthy human subjects. RESULTS: UFH showed no evidence of toxicity in mice at any dose measured. No significant changes were observed in activated partial thromboplastin time (aPTT), platelet count, or frequency of minor irritant events over vehicle-only control. Human subjects showed no significant changes in aPTT time, international normalized ratio (INR), or platelet count over baseline measurements. No serious adverse events were observed. In vivo imaging in a mouse model showed a single phase clearance of UFH from the nasal cavity. After 12 hours, 3.2% of the administered UFH remained in the nasal cavity, decaying to background levels by 48 hours. CONCLUSIONS: UFH showed no toxic effects for extended daily intranasal dosing in mice as well as humans. The clearance kinetics of intranasal heparin solution from the nasal cavity indicates potentially protective levels for up to 12 hours after dosing.
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[Figure: see text].
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Elastina/administração & dosagem , Pré-Eclâmpsia/tratamento farmacológico , Fator B de Crescimento do Endotélio Vascular/administração & dosagem , Animais , Modelos Animais de Doenças , Células Endoteliais/efeitos dos fármacos , Feminino , Humanos , Gravidez , Ratos , Ratos Sprague-Dawley , Resultado do TratamentoRESUMO
Human cytomegalovirus (HCMV) tegument protein pp150 is essential for the completion of the final steps in virion maturation. Earlier studies indicated that three pp150nt (N-terminal one-third of pp150) conformers cluster on each triplex (Tri1, Tri2A and Tri2B), and extend towards small capsid proteins atop nearby major capsid proteins, forming a net-like layer of tegument densities that enmesh and stabilize HCMV capsids. Based on this atomic detail, we designed several peptides targeting pp150nt. Our data show significant reduction in virus growth upon treatment with one of these peptides (pep-CR2) with an IC50 of 1.33 µM and no significant impact on cell viability. Based on 3D modeling, pep-CR2 specifically interferes with the pp150-capsid binding interface. Cells pre-treated with pep-CR2 and infected with HCMV sequester pp150 in the nucleus, indicating a mechanistic disruption of pp150 loading onto capsids and subsequent nuclear egress. Furthermore, pep-CR2 effectively inhibits mouse cytomegalovirus (MCMV) infection in cell culture, paving the way for future animal testing. Combined, these results indicate that CR2 of pp150 is amenable to targeting by a peptide inhibitor, and can be developed into an effective antiviral.
Assuntos
Proteínas do Capsídeo/ultraestrutura , Fosfoproteínas/metabolismo , Fosfoproteínas/fisiologia , Proteínas da Matriz Viral/metabolismo , Proteínas da Matriz Viral/fisiologia , Animais , Capsídeo , Proteínas do Capsídeo/metabolismo , Microscopia Crioeletrônica/métodos , Citomegalovirus/genética , Citomegalovirus/metabolismo , Citomegalovirus/patogenicidade , Infecções por Citomegalovirus/metabolismo , Humanos , Camundongos , Muromegalovirus/metabolismo , Muromegalovirus/patogenicidade , Fosfoproteínas/ultraestrutura , Proteínas da Matriz Viral/ultraestrutura , Vírion , Montagem de VírusRESUMO
Elastin-like polypeptides (ELPs) are bioengineered proteins that have a unique physical property, a thermally triggered inverse phase transition, that can be exploited for drug delivery. ELP-fusion proteins can be used as soluble biologics, thermally targeted drug carriers, self-assembling nanoparticles, and slow-release drug depots. Because of their unique physical characteristics and versatility for delivery of nearly any type of therapeutic, ELP-based drug delivery systems represent a promising platform for biologics development.
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Elastina , Peptídeos , Sistemas de Liberação de Medicamentos , HumanosRESUMO
Patients with chronic kidney disease (CKD) have a high cardiovascular mortality. CKD and heart failure (HF) coexist in up to 50% of patients, and both associate with inflammation. We aimed to define the cardiac phenotype of a novel swine model of CKD and test the hypothesis that inflammation of renal origin propels the development of precursors of HF in CKD. CKD was induced in 14 pigs, which were followed for 14 wk. Renal (multidetector computed tomography) and cardiac (echocardiography) hemodynamics were quantified before and 8 wk after single intrarenal administration of placebo or a biopolymer-fused peptide inhibitor of NF-κB that blocks NF-κB activity and decreases inflammatory activity (SynB1-ELP-p50i). Blood was collected to quantify cytokines (TNF-α, monocyte chemoattractant protein-1, and interleukins), markers of inflammation (C-reactive protein), and biomarkers of HF (atrial and brain natriuretic peptides). Pigs were then euthanized, and kidneys and hearts were studied ex vivo. Normal pigs were used as time-matched controls. Renal dysfunction in CKD was accompanied by cardiac hypertrophy and fibrosis, diastolic dysfunction, increased renal and cardiac expression of TNF-α, monocyte chemoattractant protein-1, and interleukins, canonical and noncanonical mediators of NF-κB signaling, circulating inflammatory factors, and biomarkers of HF. Notably, most of these changes were improved after intrarenal SynB1-SynB1-ELP-p50i, although cardiac inflammatory signaling remained unaltered. The translational traits of this model support its use as a platform to test novel technologies to protect the kidney and heart in CKD. A targeted inhibition of renal NF-κB signaling improves renal and cardiac function, suggesting an inflammatory renal-cardio axis underlying early HF pathophysiology in CKD.NEW & NOTEWORTHY Chronic kidney disease (CKD) is a progressive disorder with high cardiovascular morbidity and mortality. This work supports the role of inflammatory cytokines of renal origin in renal-cardio pathophysiology in CKD and that the heart may be a target. Furthermore, it supports the feasibility of a new strategy in a translational fashion, using targeted inhibition of renal NF-κB signaling to offset the development of cardiac injury in CKD.
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Cardiopatias/etiologia , Rim/metabolismo , NF-kappa B/metabolismo , Insuficiência Renal Crônica/metabolismo , Animais , Apoptose , Biomarcadores/sangue , Citocinas/genética , Citocinas/metabolismo , Dieta Hiperlipídica , Feminino , Fibrose/etiologia , Fibrose/patologia , Cardiopatias/metabolismo , Rim/patologia , Masculino , SuínosRESUMO
Vascular Endothelial Growth Factor (VEGF), a key mediator of angiogenesis and vascular repair, is reduced in chronic ischemic renal diseases, leading to microvascular rarefaction and deterioration of renal function. We developed a chimeric fusion of human VEGF-A121 with the carrier protein Elastin-like Polypeptide (ELP-VEGF) to induce therapeutic angiogenesis via targeted renal VEGF therapy. We previously showed that ELP-VEGF improves renal vascular density, renal fibrosis, and renal function in swine models of chronic renal diseases. However, VEGF is a potent cytokine that induces angiogenesis and increases vascular permeability, which could cause undesired off-target effects or be deleterious in a patient with a solid tumor. Therefore, the current study aims to define the toxicological profile of ELP-VEGF and assess its risk for exacerbating tumor progression and vascularity using rodent models. A dose escalating toxicology assessment of ELP-VEGF was performed by administering a bolus intravenous injection at doses ranging from 0.1 to 200 mg/kg in Sprague Dawley (SD) rats. Blood pressure, body weight, and glomerular filtration rate (GFR) were quantified longitudinally, and terminal blood sampling and renal vascular density measurements were made 14 days after treatment. Additionally, the effects of a single administration of ELP-VEGF (0.1-10 mg/kg) on tumor growth rate, mass, and vascular density were examined in a mouse model of breast cancer. At doses up to 200 mg/kg, ELP-VEGF had no effect on body weight, caused no changes in plasma or urinary markers of renal injury, and did not induce renal fibrosis or other histopathological findings in SD rats. At the highest doses (100-200 mg/kg), ELP-VEGF caused an acute, transient hypotension (30 min), increased GFR, and reduced renal microvascular density 14 days after injection. In a mouse tumor model, ELP-VEGF did not affect tumor growth rate or tumor mass, but analysis of tumor vascular density by micro-computed tomography (µCT) revealed significant, dose dependent increases in tumor vascularity after ELP-VEGF administration. ELP-VEGF did not induce toxicity in the therapeutic dosing range, and doses one hundred times higher than the expected maximum therapeutic dose were needed to observe any adverse signs in rats. In breast tumor-bearing mice, ELP-VEGF therapy induced a dose-dependent increase in tumor vascularity, demanding caution for potential use in a patient suffering from kidney disease but with known or suspected malignancy.
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Produtos Biológicos/farmacologia , Neoplasias da Mama/irrigação sanguínea , Elastina/genética , Neovascularização Patológica/induzido quimicamente , Proteínas Recombinantes de Fusão/farmacologia , Insuficiência Renal Crônica/tratamento farmacológico , Fator A de Crescimento do Endotélio Vascular/genética , Animais , Produtos Biológicos/metabolismo , Pressão Sanguínea/efeitos dos fármacos , Peso Corporal/efeitos dos fármacos , Neoplasias da Mama/diagnóstico por imagem , Neoplasias da Mama/patologia , Permeabilidade Capilar/efeitos dos fármacos , Modelos Animais de Doenças , Elastina/metabolismo , Feminino , Expressão Gênica , Taxa de Filtração Glomerular/efeitos dos fármacos , Xenoenxertos , Humanos , Hipotensão/induzido quimicamente , Hipotensão/diagnóstico por imagem , Hipotensão/fisiopatologia , Camundongos , Mimetismo Molecular , Neovascularização Patológica/diagnóstico por imagem , Neovascularização Patológica/patologia , Neovascularização Fisiológica/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Insuficiência Renal Crônica/diagnóstico por imagem , Insuficiência Renal Crônica/metabolismo , Insuficiência Renal Crônica/fisiopatologia , Suínos , Testes de Toxicidade Crônica , Fator A de Crescimento do Endotélio Vascular/metabolismo , Microtomografia por Raio-XRESUMO
BACKGROUND: Chronic renovascular disease (RVD) can lead to a progressive loss of renal function, and current treatments are inefficient. We designed a fusion of vascular endothelial growth factor (VEGF) conjugated to an elastin-like polypeptide (ELP) carrier protein with an N-terminal kidney-targeting peptide (KTP). We tested the hypothesis that KTP-ELP-VEGF therapy will effectively recover renal function with an improved targeting profile. Further, we aimed to elucidate potential mechanisms driving renal recovery. METHODS: Unilateral RVD was induced in 14 pigs. Six weeks later, renal blood flow (RBF) and glomerular filtration rate (GFR) were quantified by multidetector CT imaging. Pigs then received a single intrarenal injection of KTP-ELP-VEGF or vehicle. CT quantification of renal hemodynamics was repeated 4 weeks later, and then pigs were euthanized. Ex vivo renal microvascular (MV) density and media-to-lumen ratio, macrophage infiltration, and fibrosis were quantified. In parallel, THP-1 human monocytes were differentiated into naïve macrophages (M0) or inflammatory macrophages (M1) and incubated with VEGF, KTP-ELP, KTP-ELP-VEGF, or control media. The mRNA expression of macrophage polarization and angiogenic markers was quantified (qPCR). RESULTS: Intrarenal KTP-ELP-VEGF improved RBF, GFR, and MV density and attenuated MV media-to-lumen ratio and renal fibrosis compared to placebo, accompanied by augmented renal M2 macrophages. In vitro, exposure to VEGF/KTP-ELP-VEGF shifted M0 macrophages to a proangiogenic M2 phenotype while M1s were nonresponsive to VEGF treatment. CONCLUSIONS: Our results support the efficacy of a new renal-specific biologic construct in recovering renal function and suggest that VEGF may directly influence macrophage phenotype as a possible mechanism to improve MV integrity and function in the stenotic kidney.
Assuntos
Proteínas Recombinantes de Fusão/administração & dosagem , Recuperação de Função Fisiológica/efeitos dos fármacos , Obstrução da Artéria Renal/tratamento farmacológico , Fator A de Crescimento do Endotélio Vascular/administração & dosagem , Animais , Aterosclerose/complicações , Aterosclerose/tratamento farmacológico , Modelos Animais de Doenças , Elastina/administração & dosagem , Elastina/genética , Feminino , Taxa de Filtração Glomerular/efeitos dos fármacos , Taxa de Filtração Glomerular/fisiologia , Humanos , Rim/irrigação sanguínea , Rim/efeitos dos fármacos , Rim/fisiologia , Masculino , Microvasos/efeitos dos fármacos , Microvasos/fisiologia , Peptídeos/administração & dosagem , Peptídeos/genética , Proteínas Recombinantes de Fusão/genética , Obstrução da Artéria Renal/etiologia , Circulação Renal/efeitos dos fármacos , Sus scrofa , Fator A de Crescimento do Endotélio Vascular/genéticaRESUMO
Inflammation is a major determinant for the progression of chronic kidney disease (CKD). NF-κB is a master transcription factor upregulated in CKD that promotes inflammation and regulates apoptosis and vascular remodeling. We aimed to modulate this pathway for CKD therapy in a swine model of CKD using a peptide inhibitor of the NF-κB p50 subunit (p50i) fused to a protein carrier [elastin-like polypeptide (ELP)] and equipped with a cell-penetrating peptide (SynB1). We hypothesized that intrarenal SynB1-ELP-p50i therapy would inhibit NF-κB-driven inflammation and induce renal recovery. CKD was induced in 14 pigs. After 6 wk, pigs received single intrarenal SynB1-ELP-p50i therapy (10 mg/kg) or placebo (n = 7 each). Renal hemodynamics were quantified in vivo using multidetector computed tomography before and 8 wk after treatment. Pigs were then euthanized. Ex vivo experiments were performed to quantify renal activation of NF-κB, expression of downstream mediators of NF-κB signaling, renal microvascular density, inflammation, and fibrosis. Fourteen weeks of CKD stimulated NF-κB signaling and downstream mediators (e.g., TNF-α, monocyte chemoattractant protein-1, and IL-6) accompanying loss of renal function, inflammation, fibrosis, and microvascular rarefaction versus controls. All of these were improved after SynB1-ELP-p50i therapy, accompanied by reduced circulating inflammatory cytokines as well, which were evident up to 8 wk after treatment. Current treatments for CKD are largely ineffective. Our study shows the feasibility of a new treatment to induce renal recovery by offsetting inflammation at a molecular level. It also supports the therapeutic potential of targeted inhibition of the NF-κB pathway using novel drug delivery technology in a translational model of CKD.
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Rim/efeitos dos fármacos , NF-kappa B/antagonistas & inibidores , Insuficiência Renal Crônica/tratamento farmacológico , Animais , Modelos Animais de Doenças , Sistemas de Liberação de Medicamentos , Fibrose/tratamento farmacológico , Fibrose/metabolismo , Fibrose/patologia , Rim/metabolismo , Rim/patologia , Circulação Renal/efeitos dos fármacos , Insuficiência Renal Crônica/metabolismo , Insuficiência Renal Crônica/patologia , Transdução de Sinais/efeitos dos fármacos , SuínosRESUMO
Ischemic strokes occur when a major cerebral artery or its branches are occluded, resulting in activation of inflammatory processes that cause secondary tissue injury, breakdown of the blood-brain barrier, edema or hemorrhage. Treatments that inhibit inflammatory processes may thus be highly beneficial. A key regulator of the inflammatory process is the nuclear factor kappa B (NF-κB) pathway. In its active form, NF-κB regulates expression of proinflammatory and proapoptotic genes. The molecules that interact with NF-κB, and the subunits that compose NF-κB itself, represent therapeutic targets that can be modulated to decrease inflammation. This review focuses on our current understanding of the NF-κB pathway and the potential benefits of inhibiting NF-κB in ischemia-reperfusion injury of the brain.
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Isquemia Encefálica , AVC Isquêmico , Traumatismo por Reperfusão , Acidente Vascular Cerebral , Isquemia Encefálica/tratamento farmacológico , Humanos , NF-kappa B , Traumatismo por Reperfusão/tratamento farmacológico , Acidente Vascular Cerebral/tratamento farmacológicoRESUMO
The goals of this study are to evaluate the ability of the multicomponent collagen-elastin-like polypeptide (ELP)-Bioglass scaffolds to support osteogenesis of rat mesenchymal stem cells (rMSCs), demonstrate in vivo biocompatibility by subcutaneous implantation in Sprague-Dawley rats, monitor degradation noninvasively, and finally assess the scaffold's ability in healing critical-sized cranial bone defects. The collagen-ELP-Bioglass scaffold supports the in vitro osteogenic differentiation of rMSCs over a 3 week culture period. The cellular (rMSC-containing) or acellular scaffolds implanted in the subcutaneous pockets of rats do not cause any local or systemic toxic effects or tumors. The real-time monitoring of the fluorescently labeled scaffolds by IVIS reveals that the scaffolds remain at the site of implantation for up to three weeks, during which they degrade gradually. Micro-CT analysis shows that the bilateral cranial critical-sized defects created in rats lead to greater bone regeneration when filled with cellular scaffolds. Bone mineral density and bone microarchitectural parameters are comparable among different scaffold groups, but the histological analysis reveals increased formation of high-quality mature bone in the cellular group, while the acellular group has immature bone and organized connective tissue. These results suggest that the rMSC-seeded collagen-ELP-Bioglass composite scaffolds can aid in better bone healing process.
Assuntos
Elastina , Osteogênese , Animais , Regeneração Óssea , Diferenciação Celular , Cerâmica , Colágeno , Peptídeos , Ratos , Ratos Sprague-Dawley , Engenharia Tecidual , Alicerces TeciduaisRESUMO
Preeclampsia is a hypertensive disorder of pregnancy that causes significant acute and long-term risk to the mother and the baby. The multifaceted maternal syndrome is driven by overproduction of circulating anti-angiogenic factors, widespread inflammation, and endothelial dysfunction. Nuclear factor-κB (NF-κB) is a transcription factor that plays a central role in the inflammatory response. Its activity is increased in the preeclamptic placenta, and it promotes the systemic endothelial dysfunction present in preeclampsia. There is an acute need for new therapeutics targeted to the causative pathways of preeclampsia. Our group has developed a drug delivery system based on the bioengineered protein ELP (elastin-like polypeptide) that is capable of stabilizing therapeutics in the maternal circulation and preventing their placental transfer. Here we used the ELP carrier system to deliver a peptide known to inhibit the NF-κB pathway. This polypeptide, containing a cell-penetrating peptide and an NF-κB inhibitory peptide derived from the p50 nuclear localization sequence (abbreviated SynB1-ELP-p50i), blocked NF-κB activation and prevented TNF-α (tumor necrosis factor alpha)-induced endothelin production in vitro. Fusion of the p50i peptide to the SynB1-ELP carrier slowed its plasma clearance and prevented its placental transfer in pregnant rats, resulting in increased deposition in the maternal kidney, liver, and placenta relative to the free peptide. When administered in a rat model of placental ischemia, SynB1-ELP-p50i partially ameliorated placental ischemia-induced hypertension and reduced placental TNF-α levels with no signs of toxicity. These data support the continued development of ELP-delivered NF-κB inhibitors as maternally sequestered anti-inflammatory agents for preeclampsia therapy.
Assuntos
Biopolímeros/administração & dosagem , Sistemas de Liberação de Medicamentos , NF-kappa B/antagonistas & inibidores , Peptídeos/uso terapêutico , Pré-Eclâmpsia/tratamento farmacológico , Animais , Modelos Animais de Doenças , Feminino , Peptídeos/administração & dosagem , Pré-Eclâmpsia/sangue , Gravidez , Ratos , Fator de Necrose Tumoral alfa/sangueRESUMO
Elastin-like polypeptides (ELP) are versatile protein biopolymers used in drug delivery due to their modular nature, allowing fusion of therapeutics and targeting agents. We previously developed an ELP fusion with vascular endothelial growth factor (VEGF) and demonstrated its therapeutic efficacy in translational swine models of renovascular disease and chronic kidney disease. The goal of the current work was to refine renal targeting and reduce off-target tissue deposition of ELP-VEGF. The ELP-VEGF fusion protein was modified by adding a kidney-targeting peptide (KTP) to the N-terminus. All control proteins (ELP, KTP-ELP, ELP-VEGF, and KTP-ELP-VEGF) were also produced to thoroughly assess the effects of each domain on in vitro cell binding and activity and in vivo pharmacokinetics and biodistribution. KTP-ELP-VEGF was equipotent to ELP-VEGF and free VEGF in vitro in the stimulation of primary glomerular microvascular endothelial cell proliferation, tube formation, and extracellular matrix invasion. The contribution of each region of the KTP-ELP-VEGF protein to the cell binding specificity was assayed in primary human renal endothelial cells, tubular epithelial cells, and podocytes, demonstrating that the VEGF domain induced binding to endothelial cells and the KTP domain increased binding to all renal cell types. The pharmacokinetics and biodistribution of KTP-ELP-VEGF and all control proteins were determined in SKH-1 Elite hairless mice. The addition of KTP to ELP slowed its in vivo clearance and increased its renal deposition. Furthermore, addition of KTP redirected ELP-VEGF, which was found at high levels in the liver, to the kidney. Intrarenal histology showed similar distribution of all proteins, with high levels in blood vessels and tubules. The VEGF-containing proteins also accumulated in punctate foci in the glomeruli. These studies provide a thorough characterization of the effects of a kidney-targeting peptide and an active cytokine on the biodistribution of these novel biologics. Furthermore, they demonstrate that renal specificity of a proven therapeutic can be improved using a targeting peptide.
RESUMO
Chronic kidney disease (CKD) universally associates with renal microvascular rarefaction and inflammation, but whether a link exists between these 2 processes is unclear. We designed a therapeutic construct of VEGF (vascular endothelial growth factor) fused to an ELP (elastin-like polypeptide) carrier and show that it improves renal function in experimental renovascular disease. We test the hypothesis that ELP-VEGF therapy will improve CKD, and that recovery will be driven by decreasing microvascular rarefaction partly via modulation of macrophage phenotype and inflammation. CKD was induced in 14 pigs, which were observed for 14 weeks. At 6 weeks, renal blood flow and filtration were quantified using multidetector computed tomography, and then pigs received single intrarenal ELP-VEGF or placebo (n=7 each). Renal function was quantified again 4 and 8 weeks later. Pigs were euthanized and renal microvascular density, angiogenic and inflammatory markers, fibrosis, macrophage infiltration, and phenotype were quantified. Loss of renal hemodynamics in CKD was progressively recovered by ELP-VEGF therapy, accompanied by improved renal microvascular density, fibrosis, and expression of inflammatory mediators. Although renal macrophage infiltration was similar in both CKD groups, ELP-VEGF therapy distinctly shifted their phenotype from proinflammatory M1 to VEGF-expressing M2. Our study unravels potential mechanisms and feasibility of a new strategy to offset progression of CKD using drug-delivery technologies. The results indicate that renal recovery after ELP-VEGF therapy was largely driven by modulation of renal macrophages toward VEGF-expressing M2 phenotype, restoring VEGF signaling and sustaining improvement of renal function and microvascular integrity in CKD.