ITLN1 modulates invasive potential and metabolic reprogramming of ovarian cancer cells in omental microenvironment.

Advanced ovarian cancer usually spreads to the omentum. However, the omental cell-derived molecular determinants modulating its progression have not been thoroughly characterized. Here, we show that circulating ITLN1 has prognostic significance in patients with advanced ovarian cancer. Further studies demonstrate that ITLN1 suppresses lactotransferrin's effect on ovarian cancer cell invasion potential and proliferation by decreasing MMP1 expression and inducing a metabolic shift in metastatic ovarian cancer cells. Additionally, ovarian cancer-bearing mice treated with ITLN1 demonstrate marked decrease in tumor growth rates. These data suggest that downregulation of mesothelial cell-derived ITLN1 in the omental tumor microenvironment facilitates ovarian cancer progression.

The effect of omentin-1 on the proliferation and apoptosis of colon cancer stem cells and the potential mechanism.

PURPOSE: To investigate the effect of omentin-1 on the proliferation and apoptosis of colon cancer stem cells and the underlying mechanism. METHODS: Colon cancer stem cells were obtained by indirect immune-magnetic beads cultured in serum-free medium, and identified by spheres formation assay, differentiation assay and flow cytometry. Colon cancer stem cells were divided into the control group, the omentin-1 group (1 µg/ml omentin-1), the omentin-2 group (2 µg/ml omentin-1), the omentin-LY group (1 µg/ml omentin-1 and 50 µM LY294002) and the LY group (50 µM LY294002). CCK-8 and flow cytometry were used to detect the proliferation and apoptosis, respectively. The cell proliferation was evaluated at 0, 1, 6, 24 and 48 hrs after the intervention by omentin-1. Western blot was performed to measure the effect of different concentrations of omentin-1 on phosphorylated Akt. RESULTS: The colon cancer stem cells were successfully sorted, and the content of CD133+ in colon cancer stem cells reached 80.3%. Omentin-1 inhibited the proliferation and promoted apoptosis of colon cancer stem cells in a dose and time-dependent manner, which could be strengthened by the PI3K/Akt inhibitor. CONCLUSION: Omentin-1 could inhibit the proliferation and promote apoptosis of colon cancer stem cells in vitro via the PI3K/Akt pathway.

Additive effects of the combined expression of soluble forms of GAS1 and PTEN inhibiting glioblastoma growth.

The overexpression of GAS1 (Growth Arrest Specific 1) in glioma cells induces cell cycle arrest and apoptosis. We previously demonstrated that the apoptotic process set off by GAS1 is caused by its capacity to inhibit the Glial cell-derived neurotrophic factor (GDNF)-mediated intracellular survival signaling pathway. Whereas on the other hand, PTEN is a tumor suppressor, inactive in many tumors, and both GAS1 and PTEN inhibit the PI3K/AKT pathway. Therefore, it is relevant to investigate the potential additive effect of the overexpression of GAS1 and PTEN on tumor growth. In particular, we employed secreted forms of both GAS1 (tGAS1) and PTEN (PTEN-LONG, or PTEN-L) and tested their combined effect on glioma cells. We observed that the co-expression of both the proteins inhibited the growth of U-87 MG human glioblastoma cells more effectively than when independently expressed, and decreased the activity of both AKT and ERK1/2. Interestingly, the combination of the soluble forms was always the most effective treatment. To improve the transfer of tGAS1 and PTEN-L, we employed a lentiviral vector with a p2A peptide-enabled dual expression system that allowed the generation of the two proteins using a single promoter (CMV), in equimolar amounts. The viral vector reduced the growth of U-87 MG cells in vitro and had a striking effect in inhibiting their proliferation after inoculating it into the immunosuppressed mice. The present results support a potential adjuvant role for the combined use of tGAS1 and PTEN-L in the treatment of glioblastoma.

Neuritin attenuates early brain injury in rats after experimental subarachnoid hemorrhage.

OBJECTIVES: Early brain injury (EBI) is central to the pathological progress of subarachnoid hemorrhage (SAH). In this study, we determined if neuritin protects the brain against EBI in rats and discussed the role of apoptosis pathway mediated by endoplasmic reticulum stress in this neuroprotective route. METHODS: A total of 96 male Sprague Dawley rats were divided into control, sham, SAH and SAH + neuritin groups. The rat SAH model was induced by injection 0.3 mL of nonheparinized arterial blood into the prechiasmatic cistern. Mortality assay, neurological scores, brain water content measurement, Evans blue dye assay, TUNEL stain assay and Western blot analysis were performed. RESULTS: Neuritin significantly improved the neurological scores, brain water content, blood-brain barrier (BBB) and apoptosis compared with the control and sham groups within 24 h after SAH. TUNEL staining assay results demonstrated that apoptosis was ameliorated, MMP-9 expression was reduced, whereas GRP78, CHOP, caspase-12 and ASK1 levels were markedly preserved after neuritin application. CONCLUSIONS: Our study demonstrated that neuritin plays a neuroprotective role on EBI after SAH by attenuating BBB disruption, brain edema and apoptosis.

Co-delivery of GPI-anchored CCL28 and influenza HA in chimeric virus-like particles induces cross-protective immunity against H3N2 viruses.

Influenza infection typically initiates at respiratory mucosal surfaces. Induction of immune responses at the sites where pathogens initiate replication is crucial for the prevention of infection. We studied the adjuvanticity of GPI-anchored CCL28 co-incorporated with influenza HA-antigens in chimeric virus-like particles (cVLPs), in boosting strong protective immune responses through an intranasal (i.n.) route in mice. We compared the immune responses to that from influenza VLPs without CCL28, or physically mixed with soluble CCL28 at systemic and various mucosal compartments. The cVLPs containing GPI-CCL28 showed in-vitro chemotactic activity towards spleen and lung cells expressing CCR3/CCR10 chemokine receptors. The cVLPs induced antigen specific endpoint titers and avidity indices of IgG in sera and IgA in tracheal, lung, and intestinal secretions, significantly higher (4-6 fold) than other formulations. Significantly higher (3-5 fold) hemagglutination inhibition titers and high serum neutralization against H3N2 viruses were also detected with CCL28-containing VLPs compared to other groups. The CCL28-containing VLPs showed complete and 80% protection, when vaccinated animals were challenged with A/Aichi/2/1968/H3N2 (homologous) and A/Philippines/2/1982/H3N2 (heterologous) viruses, respectively. Thus, GPI-anchored CCL28 in influenza VLPs act as a strong immunostimulator at both systemic and mucosal sites, boosting significant cross-protection in animals against heterologous viruses across a large distance.

Semaphorin 7a exerts pleiotropic effects to promote breast tumor progression.

Understanding what drives breast tumor progression is of utmost importance for blocking tumor metastasis; we have identified that semaphorin 7a is a potent driver of ductal carcinoma in situ (DCIS) progression. Semaphorin 7a is a glycophosphatidylinositol membrane-anchored protein that promotes attachment and spreading in multiple cell types. Here, we show that increased expression of SEMA7A occurs in a large percentage of breast cancers and is associated with decreased overall and distant metastasis-free survival. In both in vitro and in vivo models, short hairpin-mediated silencing of SEMA7A reveals roles for semaphorin 7a in the promotion of DCIS growth, motility and invasion as well as lymphangiogenesis in the tumor microenvironment. Our studies also uncover a relationship between COX-2 and semaphorin 7a expression and suggest that semaphorin 7a promotes tumor cell invasion on collagen and lymphangiogenesis via activation of ß1-integrin receptor. Our results suggest that semaphorin 7a may be novel target for blocking breast tumor progression.

Preclinical and first-in-human evaluation of PRX-105, a PEGylated, plant-derived, recombinant human acetylcholinesterase-R.

PRX-105 is a plant-derived recombinant version of the human 'read-through' acetylcholinesterase splice variant (AChE-R). Its active site structure is similar to that of the synaptic variant, and it displays the same affinity towards organophosphorus (OP) compounds. As such, PRX-105 may serve as a bio-scavenger for OP pesticides and chemical warfare agents. To assess its potential use in prophylaxis and treatment of OP poisoning we conducted several preliminary tests, reported in this paper. Intravenous (IV) PRX-105 was administered to mice either before or after exposure to an OP toxin. All mice who received an IV dose of 50nmol/kg PRX-105, 2min before being exposed to 1.33×LD50 and 1.5×LD50 of toxin and 10min after exposure to 1.5×LD50 survived. The pharmacokinetic and toxicity profiles of PRX-105 were evaluated in mice and mini-pigs. Following single and multiple IV doses (50 to 200mg/kg) no deaths occurred and no significant laboratory and histopathological changes were observed. The overall elimination half-life (t½) in mice was 994 (±173) min. Additionally, a first-in-human study, to assess the safety, tolerability and pharmacokinetics of the compound, was conducted in healthy volunteers. The t½ in humans was substantially longer than in mice (average 26.7h). Despite the small number of animals and human subjects who were assessed, the fact that PRX-105 exerts a protective and therapeutic effect following exposure to lethal doses of OP, its favorable safety profile and its relatively long half-life, renders it a promising candidate for treatment and prophylaxis against OP poisoning and warrants further investigation.

Microdosing of a Carbon-14 Labeled Protein in Healthy Volunteers Accurately Predicts Its Pharmacokinetics at Therapeutic Dosages.

Preclinical development of new biological entities (NBEs), such as human protein therapeutics, requires considerable expenditure of time and costs. Poor prediction of pharmacokinetics in humans further reduces net efficiency. In this study, we show for the first time that pharmacokinetic data of NBEs in humans can be successfully obtained early in the drug development process by the use of microdosing in a small group of healthy subjects combined with ultrasensitive accelerator mass spectrometry (AMS). After only minimal preclinical testing, we performed a first-in-human phase 0/phase 1 trial with a human recombinant therapeutic protein (RESCuing Alkaline Phosphatase, human recombinant placental alkaline phosphatase [hRESCAP]) to assess its safety and kinetics. Pharmacokinetic analysis showed dose linearity from microdose (53 µg) [(14) C]-hRESCAP to therapeutic doses (up to 5.3 mg) of the protein in healthy volunteers. This study demonstrates the value of a microdosing approach in a very small cohort for accelerating the clinical development of NBEs.

Recombinant Salmonella-based CEACAM6 and 4-1BBL vaccine enhances T-cell immunity and inhibits the development of colorectal cancer in rats: In vivo effects of vaccine containing 4-1BBL and CEACAM6.

The present study aimed to determine the effect of recombinant Salmonella (SL3261)-based CEACAM6 and 4-1BB ligand (4-1BBL) vaccine on the development of colorectal cancer in rats and the potential immune mechanisms involved. Attenuated Salmonella typhimurium (vaccine strain)­carrying plasmids pIRES-CEACAM6, pIRES­4­1BBL and pIRES-CEACAM6-4-1BBL were constructed. The rats were administered subcutaneous injections of 1,2-dimethyl-hydrazine (DMH) once a week for 18 weeks. Eight weeks after the first injection, the rats were divided into the pIRES/SL3261, pIRES-4-1BBL/SL3261, pIRES-CEACAM6/SL3261 and pIRES-CEACAM6-4-1BBL/SL3261 groups, and fed with corresponding vaccine strains. The rats were then sacrificed, the number of colon tumors were recorded, and the Dukes' stage were evaluated. CD3, CD4, CD8, CD56, FOXP3 and CEACAM6 expression in tumor tissues was determined by immunohistochemical staining. Compared with the expression levels in the pIRES/SL3261 group, similar levels of CD3+, CD8+ and CD56+ expression were identified for the pIRES-CEACAM6/SL3261 group of rats. Additionally, a comparable number of tumors was detected in the pIRES-4-1BBL/SL3261 and pIRES-CEACAM6/SL3261 groups. By contrast, a significantly fewer number of tumors, albeit with a higher density of CD3+CD8+, CD56+ and a lower density of Foxp3+ tumor-infiltrating lymphocyte (TIL) cells was detected in the pIRES-CEACAM6-4-1BBL/SL3261 group of rats. The results indicated that vaccination with recombinant attenuated Salmonella harboring the CEACAM6 and 4-1BBL gene efficiently increased the number of CD3+CD8+ TIL and NK cells, decreased the number of FOXP3 cells and inhibited the development of DMH-induced colorectal cancer in rats.

Study on the mesothelin-specific cytotoxicity against epithelial ovarian cancer with full-length mesothelin cDNA-transduced dendritic cells.

Epithelial ovarian cancer (EOC) has the highest mortality rate among the various types of gynecological cancers. As the current therapeutic approaches are not enough, the development of more effective treatments to improve the survival of patients with EOC is urgently needed. Mesothelin (MSLN) is a cell surface glycoprotein, which is overexpressed in ovarian cancer tissues. As an immunotherapeutic approach, in this study, we investigated whether the genetically modified dendritic cells (DCs) expressing MSLN could induce cytotoxic T lymphocytes (CTLs) to produce MSLN-specific cytotoxic activity against EOCs. Here, we report that DCs transfected with full-length coding sequence of MSLN could induce MSLN-specific CTLs responses against ovarian cancer lines SKOV3 and OVCAR3 in vitro. Additionally, we identified that the death rates of ovarian cancer cells, killed by MSLN-specific CTLs, were significantly higher than the normal CTLs. Furthermore, IFN-γ production by stimulated MSLN-specific CTLs was significantly higher than that of unstimulated CTLs. This study showed that induced CTLs by DCs with full-length MSLN cDNA have effective immune response against the ovarian cancer cells, indicating that MSLN-transfected DCs vaccine has a promising prospect for the treatment of EOC.

Intravitreal delivery of human NgR-Fc decoy protein regenerates axons after optic nerve crush and protects ganglion cells in glaucoma models.

PURPOSE: Glaucoma is a major cause of vision loss due to retinal ganglion cell (RGC) degeneration. Therapeutic intervention controls increased IOP, but neuroprotection is unavailable. NogoReceptor1 (NgR1) limits adult central nervous system (CNS) axonal sprouting and regeneration. We examined NgR1 blocking decoy as a potential therapy by defining the pharmacokinetics of intravitreal NgR(310)-Fc, its promotion of RGC axonal regeneration following nerve crush, and its neuroprotective effect in a microbead glaucoma model. METHODS: Human NgR1(310)-Fc was administered intravitreally, and levels were monitored in rat vitreal humor and retina. Axonal regeneration after optic nerve crush was assessed by cholera toxin ß anterograde labeling. In a microbead model of glaucoma with increased IOP, the number of surviving and actively transporting RGCs was determined after 4 weeks by retrograde tracing with Fluro-Gold (FG) from the superior colliculus. RESULTS: After intravitreal bolus administration, the terminal half-life of NgR1(310)-Fc between 1 and 7 days was approximately 24 hours. Injection of 5 µg protein once per week after optic nerve crush injury significantly increased RGCs with regenerating axons. Microbeads delivered to the anterior chamber increased pressure, and caused 15% reduction in FG-labeled RGCs of control rats, with a 40% reduction in large diameter RGCs. Intravitreal treatment with NgR1(310)-Fc did not reduce IOP, but maintained large diameter RGC density at control levels. CONCLUSIONS: Human NgR1(310)-Fc has favorable pharmacokinetics in the vitreal space and rescues large diameter RGC counts from increased IOP. Thus, the NgR1 blocking decoy protein may have efficacy as a disease-modifying therapy for glaucoma.

Omentin functions to attenuate cardiac hypertrophic response.

Cardiac hypertrophy occurs in many obesity-related conditions. Omentin is an adipose-derived plasma protein that is downregulated under obese conditions. Here, we investigated whether omentin modulates cardiac hypertrophic responses in vivo and in vitro. Systemic administration of an adenoviral vector expressing human omentin (Ad-OMT) to wild-type (WT) mice led to the attenuation of cardiac hypertrophy, fibrosis and ERK phosphorylation induced by transverse aortic constriction (TAC) or angiotensin II infusion. In cultured cardiomyocytes, stimulation with phenylephrine (PE) led to an increase in myocyte size, which was prevented by pretreatment with human omentin protein. Pretreatment of cardiomyocytes with omentin protein also reduced ERK phosphorylation in response to PE stimulation. Ad-OMT enhanced phosphorylation of AMP-activated protein kinase (AMPK) in the heart of WT mice after TAC operation. Blockade of AMPK activation by transduction with dominant-negative mutant forms of AMPK reversed the inhibitory effect of omentin on myocyte hypertrophy and ERK phosphorylation following PE stimulation. Moreover, fat-specific transgenic mice expressing human omentin showed reduced cardiac hypertrophy and ERK phosphorylation following TAC surgery compared to littermate controls. These data suggest that omentin functions to attenuate the pathological process of myocardial hypertrophy via the activation of AMPK in the heart, suggesting that omentin may represent a target molecule for the treatment of cardiac hypertrophy.

Hypotensive effects of omentin-1 related to increased adiponectin and decreased interleukin-6 in intra-thoracic pericardial adipose tissue.

BACKGROUND: Omentin is an adipokine expressed in visceral adipose tissue (VAT). In vitro studies demonstrated that omentin induces vasorelaxation in isolated rat mesenteric arteries, and in vivo studies showed inhibition of agonist-induced increases in blood pressure, possibly mediated by nitric oxide (NO)-dependent mechanisms. METHODS: We investigated, in normotensive rats, the effects of subacute omentin-1 administration [8µg/kg, intraperitoneally (ip), once daily for 14 days] on cardiac activity, blood pressure, plasma concentration of l-citrulline (as a marker of NO production from l-arginine), and the gene expression of adiponectin, tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) in intra-thoracic pericardial adipose tissue (PAT). Electrocardiography (ECG), heart rate (HR), mean blood pressure (MBP), pulse pressure (PP) were monitored before and after treatment with omentin-1 or vehicle. RESULTS: With respect to baseline and vehicle, we found a significant decrease of MBP (p<0.005) and PP (p<0.05) after treatment with omentin-1, while ECG and HR were not modified. Omentin-1 significantly increased l-citrulline levels in plasma (p<0.05), and the gene expression of adiponectin in PAT (p<0.05). On the other hand, we found decreased gene expression of IL-6 (p<0.005), while TNF-α mRNA in PAT was not affected. CONCLUSION: We conclude that the hypotensive effects of omentin-1 could be driven by stimulated production of NO in the vascular system, possibly related to increased adiponectin and decreased IL-6 mRNA in PAT.

Phase 1 study of the antimesothelin immunotoxin SS1P in combination with pemetrexed and cisplatin for front-line therapy of pleural mesothelioma and correlation of tumor response with serum mesothelin, megakaryocyte potentiating factor, and cancer antigen 125.

BACKGROUND: The primary objective of this study was to determine the safety and maximum tolerated dose (MTD) of the antimesothelin immunotoxin SS1(dsFv)PE38 (SS1P) (a recombinant antimesothelin immunotoxin consisting of a murine antimesothelin variable antibody fragment [Fv] linked to PE38, a truncated portion of Pseudomonas exotoxin A) in combination with pemetrexed and cisplatin in chemotherapy-naive patients with advanced malignant pleural mesothelioma (MPM). Secondary objectives included tumor response, SS1P pharmacokinetics, and serum biomarkers of response. METHODS: Chemotherapy-naive patients with stage III or IV, unresectable, epithelial or biphasic MPM and normal organ functions were eligible. Pemetrexed (500 mg/m(2) on day 1) and cisplatin (75 mg/m(2) on day 1) were administered every 3 weeks for up to 6 cycles with escalating doses of SS1P administered intravenously on days 1, 3, and 5 during cycles 1 and 2. Tumor response was evaluated every 6 weeks. RESULTS: Twenty-four patients received SS1P at 4 dose levels from 25 to 55 mcg/kg. Grade 3 fatigue was dose-limiting in 1 patient at 55 mcg/kg. The MTD of SS1P was established as 45 mcg/kg. Other grade 3 toxicities associated with SS1P included hypoalbuminemia (21%), back pain (13%), and hypotension (8%). Of 20 evaluable patients, 12 (60%) had a partial response, 3 had stable disease, and 5 had progressive disease. Of 13 patients who received the MTD, 10 (77%) had a partial response, 1 had stable disease, and 2 had progressive disease. Objective radiologic responses were associated with significant decreases in serum mesothelin (P=.0030), megakaryocyte potentiating factor (P=.0005), and cancer antigen 125 (P < .0001). CONCLUSIONS: SS1P given with pemetrexed and cisplatin is safe and well tolerated and exhibits significant antitumor activity in patients with unresectable, advanced pleural mesothelioma. Serum mesothelin, megakaryocyte potentiating factor, and cancer antigen 125 levels correlated with objective tumor responses.

Human NgR-Fc decoy protein via lumbar intrathecal bolus administration enhances recovery from rat spinal cord contusion.

Axonal growth and neurological recovery after traumatic spinal cord injury (SCI) is limited by the presence of inhibitory proteins in myelin, several of which act via the NgR1 protein in neurons. A truncated soluble ligand-binding fragment of NgR1 serves as a decoy and promotes recovery in acute and chronic rodent SCI models. To develop the translational potential of these observations, we created a human sequence-derived NgR1(310)-Fc protein. This protein is active in vitro. When the human NgR1 decoy is administered by continuous intracerebroventricular infusion to rats with a spinal contusion injury at doses of 0.09-0.53 mg/kg/d, neurological recovery is improved. Effective doses double the percentage of rats able to bear weight on their hindlimbs. Next, we considered the half-life and distribution of NgR1(310)-Fc after bolus delivery to the lumbar intrathecal space. The protein is found throughout the neuraxis and has a tissue half-life of approximately 2 days in the rat, and 5 days in the nonhuman primate. At an intermittent, once every 4 day, lumbar bolus dosing schedule of 0.14 mg/kg/d, NgR1(310)-Fc promoted locomotor rat recovery from spinal cord contusion at least as effectively as continuous infusion in open field and grid walking tasks. Moreover, the intermittent lumbar NgR1(310)-Fc treatment increased the growth of raphespinal axons into the lumbar spinal cord after injury. Thus, human NgR1(310)-Fc provides effective treatment for recovery from traumatic SCI in this preclinical model with a simplified administration regimen that facilitates clinical testing.

Prediction of protein degradation during vibrating mesh nebulization via a high throughput screening method.

Maintaining the integrity of biopharmaceuticals is a major requirement for successful pulmonary delivery by nebulization. Sparing laborious nebulization tests, this study aimed to demonstrate the feasibility of a high throughput, material saving surrogate method to predict protein stability after nebulization. Detrimental conditions during nebulization with a PARI eFlow® vibrating mesh nebulizer were mimicked by vigorous agitation at elevated temperatures. Comparing the effect of several different excipients on the stability of the protein SM101 after nebulization and after the surrogate method revealed an excellent correlation regarding SM101 aggregation (R(2)=0.97). Design of experiment was used to develop an inhalable formulation of SM101 based entirely on the new surrogate method. Two lead formulation candidates were selected based on their predicted stability profile. The conservation of full SM101 stability and activity after nebulization was confirmed for an AKITA(2) vibrating mesh nebulizer. This study demonstrated that biopharmaceutical formulation development for nebulization is feasible by means of imitating nebulizer related detrimental factors, allowing an accelerated and more economic formulation development.

That's cool!--Nebulization of thermolabile proteins with a cooled vibrating mesh nebulizer.

Despite contrary reports, heating inside the medication reservoir was observed for several vibrating mesh nebulizers, which may be detrimental when nebulizing biopharmaceuticals. In this study we evaluated different strategies to reduce reservoir heating during nebulization with a PARI eFlow® regarding cooling efficiency, impact on nebulizer performance and on protein stability after nebulization. Passive cooling was achieved by solution pre-cooling, overcharging of the reservoir with 1 mL additional solution or intermittent nebulization. Active cooling was realized with a micro Peltier element attached to the nebulizer reservoir. Passive cooling was most effective when the reservoir was overcharged with pre-cooled solution reducing the average reservoir temperature (TRES AVG) by 8.4°C. Active cooling enabled nebulization at a constant reservoir temperature (TRES) as low as 15°C. TRES manipulation had a linear impact on nebulizer performance. While the output rate decreased with decreasing TRES, the inhalable fraction increased resulting in an inhalable aerosol rate constant over a large TRES range. The effect on protein stability depended on the susceptibility to thermal stress and was predicted by Tm values. For lactic dehydrogenase and SM101, both exhibiting a Tm below 60°C, cooling was protective in increasing the residual activity and reducing protein aggregation. A more thermostable IgG1 did not benefit from cooled nebulization. Nebulizer cooling is a prerequisite to retain the activity and stability of thermolabile proteins during vibrating mesh nebulization. It is best achieved by micro Peltier based active cooling or by simple passive cooling strategies.

A novel adipocytokine, omentin, inhibits agonists-induced increases of blood pressure in rats.

Omentin is a recently identified adipocytokine, and we previously demonstrated that omentin played anti-inflammatory roles in vascular endothelial and smooth muscle cells. We also demonstrated that omentin induced vasodilation in rat isolated blood vessels. However, effects of omentin on blood pressure (BP) are not determined. Here, we examined whether intravenously injected omentin acutely alters BP of Wistar rats. Omentin (0.06-18 µg/kg) alone did not alter BP of Wistar rats. On the other hand, omentin (18 µg/kg) significantly inhibited noradrenaline (NA; 2 µg/kg)-induced increases in systolic BP and mean BP. Omentin (18 µg/kg) significantly inhibited angiotensin II (1 µg/kg)-induced increases in diastolic BP. Omentin (18 µg/kg) significantly inhibited dimorpholamine (3 mg/kg)-induced increases in diastolic BP. Omentin (18 µg/kg) failed to inhibit the NA (0.02-2 µg/kg)-induced increases of systolic BP in the nitric oxide (NO) synthase inhibitor, N(G)-nitro-l-arginine methyl ester (80 mg/kg, 1 day)-treated Wistar rats. In summary, we for the first time demonstrated that omentin inhibited agonists-induced increases in BP. The effect of omentin was suggested to be mediated likely via NO-dependent mechanism.

Transduction of skeletal muscles with common reporter genes can promote muscle fiber degeneration and inflammation.

Recombinant adeno-associated viral vectors (rAAV vectors) are promising tools for delivering transgenes to skeletal muscle, in order to study the mechanisms that control the muscle phenotype, and to ameliorate diseases that perturb muscle homeostasis. Many studies have employed rAAV vectors carrying reporter genes encoding for ß-galactosidase (ß-gal), human placental alkaline phosphatase (hPLAP), and green fluorescent protein (GFP) as experimental controls when studying the effects of manipulating other genes. However, it is not clear to what extent these reporter genes can influence signaling and gene expression signatures in skeletal muscle, which may confound the interpretation of results obtained in experimentally manipulated muscles. Herein, we report a strong pro-inflammatory effect of expressing reporter genes in skeletal muscle. Specifically, we show that the administration of rAAV6:hPLAP vectors to the hind limb muscles of mice is associated with dose- and time-dependent macrophage recruitment, and skeletal muscle damage. Dose-dependent expression of hPLAP also led to marked activity of established pro-inflammatory IL-6/Stat3, TNFα, IKKß and JNK signaling in lysates obtained from homogenized muscles. These effects were independent of promoter type, as expression cassettes featuring hPLAP under the control of constitutive CMV and muscle-specific CK6 promoters both drove cellular responses when matched for vector dose. Importantly, the administration of rAAV6:GFP vectors did not induce muscle damage or inflammation except at the highest doses we examined, and administration of a transgene-null vector (rAAV6:MCS) did not cause damage or inflammation at any of the doses tested, demonstrating that GFP-expressing, or transgene-null vectors may be more suitable as experimental controls. The studies highlight the importance of considering the potential effects of reporter genes when designing experiments that examine gene manipulation in vivo.