Transplantation of Mesenchymal Stromal Cells Expressing the Human Preproenkephalin Gene Can Relieve Pain in a Rat Model of Neuropathic Pain.

Transgenic therapy for central neuralgia faces the problems of low expression and weak targeting and affects superficial but not deep neurons. In this study, we generated a lentivirus vector with human preproenkephalin gene (hPPE) expression driven by the transcriptional amplification strategy system (TAS) and established a primary bone marrow-derived mesenchymal stromal cell (BMSC) line stably expressing hPPE for transplantation into a rat model of neuropathic pain rat. The paw thermal withdrawal latency assay and paw mechanical withdrawal threshold assay showed that unlike control BMSCs and BMSCs with hPPE overexpression driven by the CMV or Synapsin 1 (SYN1) promoter, TAS-hPPE BMSCs had a robust and lasting analgesic effect. The TAS-hPPE BMSC-treated group exhibited higher expression of TAS-driven hPPE and a higher ratio of BMSCs in the midbrain, spinal cord and cortex then the CMV-hPPE BMSC- and SYN1-hPPE BMSC-treated groups. Moreover, we also observed that TAS-hPPE BMSCs displayed a greater tendency to differentiate into neurons and exhibit neuronal-like distribution than CMV-hPPE or SYN1-hPPE BMSCs. In conclusion, our study shows that the TAS improves BMSC transgenic therapy for neuropathic pain treatment.

Procalcitonin and MR-proAdrenomedullin combination in the etiological diagnosis and prognosis of sepsis and septic shock.

Procalcitonin and Mid-regional pro Adrenomedullin have been proposed for sepsis diagnosis, antibiotic therapy guidance and prognosis. A retrospective analysis of PCT and MR-proADM on 571 consecutive patients with sepsis diagnosis was performed. Median values were compared using the non-parametric Mann-Whitney's test. Receiver operating characteristic analysis was performed to define cutoff points for sepsis diagnosis. Pretest odds, posttest odds, and posttest probability have been calculated. Data were analyzed using Med-Calc 11.6.1.0 software. PCT resulted excellent in gram-negative, but less performant in gram-positive and fungal etiologies. MR-proADM values resulted homogenously distributed within the different microbial classes and increased significantly in septic shock. PCT highest PPV value was found to distinguish gram-negative from fungal sepsis and septic shock (>3. 57 ng/mL, PPV 0.96 and > 8.77 ng/mL, PPV 0.96, respectively). Good diagnostic accuracy was evidenced to discriminate gram-negative from gram-positive septic shock (>3.88 ng/mL PPV 0.89). Lower diagnostic accuracy was evidenced to discriminate gram-negative and gram-positive sepsis (>0.80 ng/mL, PPV 0.78) and gram-positive from fungal septic shock (>1.74 ng/mL PPV 0.75). The lowest PCT PPV (0.28) was found in gram-positive and fungal sepsis distinction. MR-proADM discriminating cut-offs were homogeneously distributed in Gram-negative and Gram-positive sepsis and were higher in septic shock, but not influenced by pathogen etiologies. MR-proADM cut-off values > 3.39 nmol/L in sepsis and >4.33 nmol/L in septic shock were associated with significant higher risk of 90-days mortality. In conclusion, PCT and MR-proADM combination represents an advantage for sepsis diagnosis and for 90-days mortality risk stratification.

proBDNF Accelerates Brain Amyloid-ß Deposition and Learning and Memory Impairment in APPswePS1dE9 Transgenic Mice.

BACKGROUND: Alzheimer's disease (AD) is pathologically known for the amyloid-ß (Aß) deposition, neurofibrillary tangles, and neuronal loss in the brain. The precursor of brain-derived neurotrophic factor (proBDNF) before proteolysis has opposing functions to its mature form in neuronal survival and neurite growth. However, the role of proBDNF in the pathogenesis of AD remains unclear. OBJECTIVE: To investigate the effects of proBDNF on neurons in vitro, and on learning and memory impairment and brain Aß production in a transgenic AD mouse model (APPswePS1dE9). METHODS: We here examined the effects of proBDNF on the viability (MTT assay) and neurite growth (morphologic measurement) of the primary neurons in vitro. After the intracerebroventricular injection of adeno-associated virus-proBDNF (AAV-proBDNF), we then investigated the learning and memory impairment (Morris water maze) and Aß deposition in the brains of the AD mice. RESULTS: The results showed that proBDNF could inhibit neuronal viability and neurite growth in vitro, enhance Aß levels, and accelerate its deposition in the brain, which was consistent with the learning and memory impairment of AD mice, likely dependent on the membrane receptor of p75NTR. CONCLUSIONS: Our findings suggest that proBDNF may exert a crucially negative effect during AD pathogenesis andprogression.

Prothymosin Alpha: An Alarmin and More...

BACKGROUND/OBJECTIVE: Prothymosin alpha (proTα) is a ubiquitous polypeptide first isolated by Haritos in 1984, whose role still remains partly elusive. We know that proTα acts both, intracellularly, as an anti-apoptotic and proliferation mediator, and extracellularly, as a biologic response modifier mediating immune responses similarly to molecules termed as "alarmins". Our research team pioneered the elucidation of the mechanisms underlying the observed activities of proTα. RESULTS: We were the first to demonstrate that proTα levels increase during normal and abnormal cell proliferation. We showed that proTα acts pleiotropically, inducing immunomodulatory effects on immune cell populations. We revealed that the immunoreactive region of proTα is the carboxyterminal decapeptide proTα(100-109) and both molecules stimulate innate immune responses, signaling through Toll-like receptors (TLRs), specifically TLR-4. We reported that proTα and proTα(100-109) bind on the surface of human neutrophils on sites involving TLR-4, and cell activation is complemented by cytoplasmic calcium ion influx. Further, we showed that proTα and proTα(100-109) act as adjuvants upstream of lymphocyte stimulation and, in the presence of antigen, promote the expansion of antigen-reactive effectors. Most recently, we reported that proTα(100-109) may accumulate in experimentally inflamed sites and can serve as a surrogate biomarker in severe bacterial infections, proposing that extracellular release of proTα or proTα(100- 109) alerts the immune system during conditions of danger. CONCLUSION: We, therefore, suggest that proTα, and likely proTα(100-109), act as alarmins, being important immune mediators as well as biomarkers, and could eventually become targets for new therapeutic/diagnostic approaches in immune-related diseases like cancer, inflammation, and sepsis.

Antinociceptive Effect of Intrathecal Injection of Genetically Engineered Human Bone Marrow Stem Cells Expressing the Human Proenkephalin Gene in a Rat Model of Bone Cancer Pain.

Background. This study aimed to investigate the use of human bone marrow mesenchymal stem cells (hBMSCs) genetically engineered with the human proenkephalin (hPPE) gene to treat bone cancer pain (BCP) in a rat model. Methods. Primary cultured hBMSCs were passaged and modified with hPPE, and the cell suspensions (6 × 106) were then intrathecally injected into a rat model of BCP. Paw mechanical withdrawal threshold (PMWT) was measured before and after BCP. The effects of hPPE gene transfer on hBMSC bioactivity were analyzed in vitro and in vivo. Results. No changes were observed in the surface phenotypes and differentiation of hBMSCs after gene transfer. The hPPE-hBMSC group showed improved PMWT values on the ipsilateral side of rats with BCP from day 12 postoperatively, and the analgesic effect was reversed by naloxone. The levels of proinflammatory cytokines such as IL-1ß and IL-6 were ameliorated, and leucine-enkephalin (L-EK) secretion was augmented, in the hPPE-engineered hBMSC group. Conclusion. The intrathecal administration of BMSCs modified with the hPPE gene can effectively relieve pain caused by bone cancer in rats and might be a potentially therapeutic tool for cancer-related pain in humans.

Deletion of Nuclear Localizing Signal Attenuates Proinflammatory Activity of Prothymosin-Alpha and Enhances Its Neuroprotective Effect on Transient Ischemic Stroke.

Post-ischemic inflammation plays an important role in the progression of ischemia/reperfusion injuries. Prothymosin-α (ProT) can protect cells from necrotic death following ischemia; however, its immunostimulatory actions may counteract the neuroprotective effect. We proposed that ProTΔNLS, synthesized by deleting its nuclear localizing signal (NLS) at the C-terminal of ProT, can attenuate the immunostimulatory activity and has more salient neuroprotective effect. In this study, we examined the therapeutic effects of ProT and ProTΔNLS in a transient middle cerebral artery occlusion (tMCAO) model of rats. Rats that had sustained 90 min of tMCAO were treated with GST-vehicle, ProT, or ProTΔNLS. Therapeutic outcomes were evaluated by infarction volume assay and behavioral assessment. Changes to inflammatory mediators, including tumor necrosis factor α (TNF-α), interleukin-10 (IL-10), and myeloperoxidase (MPO) were evaluated by enzyme-linked immunosorbent assay. Activated matrix metalloproteinases 2 (MMP-2) and 9 (MMP-9) levels were evaluated by gelatin zymography. Microglial activation was identified by double-immunostaining for Iba-1 and CD68. Our results showed that while both ProT and ProTΔNLS reduce infarction volume and improve functional outcome, ProTΔNLS provides the best therapeutic outcome. ProT increases TNF-α but decreases IL-10 secretion after ischemic injury, reflecting its pro-inflammatory activity. ProTΔNLS suppresses expression of TNF-α, MPO, and activity of MMPs in ischemic brain tissue. It also suppresses activation of microglia in penumbral cortex. These data demonstrate the immunesuppressive activities of ProTΔNLS. In conclusion, ProT has pro-inflammatory effect that may counteract its neuroprotective effect. Deletion of NLS from ProT may attenuate post-ischemic inflammation and enhance the neuroprotective effects of ProT.

Prothymosin Alpha and Immune Responses: Are We Close to Potential Clinical Applications?

The thymus gland produces soluble molecules, which mediate significant immune functions. The first biologically active thymic extract was thymosin fraction V, the fractionation of which led to the isolation of a series of immunoactive polypeptides, including prothymosin alpha (proTα). ProTα displays a dual role, intracellularly as a survival and proliferation mediator and extracellularly as a biological response modifier. Accordingly, inside the cell, proTα is implicated in crucial intracellular circuits and may serve as a surrogate tumor biomarker, but when found outside the cell, it could be used as a therapeutic agent for treating immune system deficiencies. In fact, proTα possesses pleiotropic adjuvant activity and a series of immunomodulatory effects (eg, anticancer, antiviral, neuroprotective, cardioprotective). Moreover, several reports suggest that the variable activity of proTα might be exerted through different parts of the molecule. We first reported that the main immunoactive region of proTα is the carboxy-terminal decapeptide proTα(100-109). In conjunction with data from others, we also revealed that proTα and proTα(100-109) signal through Toll-like receptor 4. Although their precise molecular mechanism of action is yet not fully elucidated, proTα and proTα(100-109) are viewed as candidate adjuvants for cancer immunotherapy. Here, we present a historical overview on the discovery and isolation of thymosins with emphasis on proTα and data on some immune-related new activities of the polypeptide and smaller immunostimulatory peptides thereof. Finally, we propose a compiled scenario on proTα's mode of action, which could eventually contribute to its clinical application.

Hormonal control of T-cell development in health and disease.

The physiology of the thymus, the primary lymphoid organ in which T cells are generated, is controlled by hormones. Data from animal models indicate that several peptide and nonpeptide hormones act pleiotropically within the thymus to modulate the proliferation, differentiation, migration and death by apoptosis of developing thymocytes. For example, growth hormone and prolactin can enhance thymocyte proliferation and migration, whereas glucocorticoids lead to the apoptosis of these developing cells. The thymus undergoes progressive age-dependent atrophy with a loss of cells being generated and exported, therefore, hormone-based therapies are being developed as an alternative strategy to rejuvenate the organ, as well as to augment thymocyte proliferation and the export of mature T cells to peripheral lymphoid organs. Some hormones (such as growth hormone and progonadoliberin-1) are also being used as therapeutic agents to treat immunodeficiency disorders associated with thymic atrophy, such as HIV infection. In this Review, we discuss the accumulating data that shows the thymus gland is under complex and multifaceted hormonal control that affects the process of T-cell development in health and disease.

Functional Characterization of Human ProNGF and NGF Mutants: Identification of NGF P61SR100E as a "Painless" Lead Investigational Candidate for Therapeutic Applications.

BACKGROUND: Nerve Growth Factor (NGF) holds a great therapeutic promise for Alzheimer's disease, diabetic neuropathies, ophthalmic diseases, dermatological ulcers. However, the necessity for systemic delivery has hampered the clinical applications of NGF due to its potent pro-nociceptive action. A "painless" human NGF (hNGF R100E) mutant has been engineered. It has equal neurotrophic potency to hNGF but a lower nociceptive activity. We previously described and characterized the neurotrophic and nociceptive properties also of the hNGF P61S and P61SR100E mutants, selectively detectable against wild type hNGF. However, the reduced pain-sensitizing potency of the "painless" hNGF mutants has not been quantified. OBJECTIVES AND RESULTS: Aiming at the therapeutic application of the "painless" hNGF mutants, we report on the comparative functional characterization of the precursor and mature forms of the mutants hNGF R100E and hNGF P61SR100E as therapeutic candidates, also in comparison to wild type hNGF and to hNGF P61S. The mutants were assessed by a number of biochemical, biophysical methods and assayed by cellular assays. Moreover, a highly sensitive ELISA for the detection of the P61S-tagged mutants in biological samples has been developed. Finally, we explored the pro-nociceptive effects elicited by hNGF mutants in vivo, demonstrating an expanded therapeutic window with a ten-fold increase in potency. CONCLUSIONS: This structure-activity relationship study has led to validate the concept of developing painless NGF as a therapeutic, targeting the NGF receptor system and supporting the choice of hNGF P61S R100E as the best candidate to advance in clinical development. Moreover, this study contributes to the identification of the molecular determinants modulating the properties of the hNGF "painless" mutants.

[Micropump infusion of gonadorelin in the treatment of hypogonadotropic hypogonadism in patients with pituitary stalk interruption syndrome: cases analysis and literature review].

Two cases of hypogonadotropic hypogonadism caused by pituitary stalk interruption syndrome treated by pulse infusion of gonadorelin via micropump were reported, and their clinical features and the treatment process of pulse infusion of gonadorelin via micropump summarized. Both of the 2 patients were presented primarily with hypogonadotropic hypogonadism. After the treatment with pulse infusion of gonadorelin via micropump, their syndrome of androgen deficiency improved and the gonadotropin levels promoted at the end of 12 weeks' follow-up. Pulse infusion of gonadorelin via micropump is an alternative to treat hypogonadotropic hypogonadism caused by pituitary stalk interruption syndrome.

Latency can be conferred to a variety of cytokines by fusion with latency-associated peptide from TGF-ß.

OBJECTIVES: Targeting cytokines to sites of disease has clear advantages because it increases their therapeutic index. We designed fusion proteins of the latent-associated peptide (LAP) derived from TGF-ß with various cytokines via a matrix metalloproteinase (MMP) cleavage site. This design confers latency, increased half-life and targeting to sites of inflammation. The aim of this study is to determine whether this approach can be applied to cytokines of different molecular structures and sizes. METHODS: Mature cytokines cloned downstream of LAP and a MMP cleavage site were expressed in 293T cells and assessed for latency and biological activity by Western blotting and bioassay. RESULTS: We demonstrate here that fusion proteins of TGF-ß, erythropoietin, IL-1ra, IL-10, IL-4, BMP-7, IGF1 and IL-17 were rendered latent by fusion to LAP, requiring cleavage to become active in respective bioassays. As further proof of principle, we also show that delivery of engineered TGF-ß can inhibit experimental autoimmune encephalomyelitis and that this approach can be used to efficiently deliver cytokines to the brain and spinal cord in mice with this disease. CONCLUSIONS: The latent cytokine approach can be successfully applied to a range of molecules, including cytokines of different molecular structure and mass, growth factors and a cytokine antagonist.

Central role of ß-catenin in anticancer effects of cardiac hormones.

BACKGROUND: ß-Catenin causes malignant growth of colonic, pancreatic and renal cancer. Four cardiac hormones, namely atrial natriuretic peptide (ANP), vessel dilator, long-acting natriuretic peptide (LANP) and kaliuretic peptide eliminate up to 80% of human pancreatic carcinomas growing in mice. MATERIALS AND METHODS: Four cardiac hormones were evaluated for their ability to reduce the expression of human ß-catenin, measured by enzyme-linked immunosorbent assay (ELISA) in human colorectal, pancreatic and renal cancer cells. RESULTS: Vessel dilator, LANP, kaliuretic peptide, and ANP, over a concentration range of 100 pM to 10 µM, maximally reduced expression of ß-catenin in human colorectal cancer cells by 78%, 71%, 69%, and 83%, respectively. Vessel dilator, LANP, kaliuretic peptide, and ANP reduced ß-catenin expression in human pancreatic cancer cells by 76%, 66%, 72%, and 88%, and by 64%, 54%, 58% and 73%, in human renal cancer cells, respectively. CONCLUSION: Part of the anticancer action of these four cardiac hormones is a potent inhibition of ß-catenin.

Prothymosin alpha: a ubiquitous polypeptide with potential use in cancer diagnosis and therapy.

The thymus is a central lymphoid organ with crucial role in generating T cells and maintaining homeostasis of the immune system. More than 30 peptides, initially referred to as "thymic hormones," are produced by this gland. Although the majority of them have not been proven to be thymus-specific, thymic peptides comprise an effective group of regulators, mediating important immune functions. Thymosin fraction five (TFV) was the first thymic extract shown to stimulate lymphocyte proliferation and differentiation. Subsequent fractionation of TFV led to the isolation and characterization of a series of immunoactive peptides/polypeptides, members of the thymosin family. Extensive research on prothymosin α (proTα) and thymosin α1 (Tα1) showed that they are of clinical significance and potential medical use. They may serve as molecular markers for cancer prognosis and/or as therapeutic agents for treating immunodeficiencies, autoimmune diseases and malignancies. Although the molecular mechanisms underlying their effect are yet not fully elucidated, proTα and Tα1 could be considered as candidates for cancer immunotherapy. In this review, we will focus in principle on the eventual clinical utility of proTα, both as a tumor biomarker and in triggering anticancer immune responses. Considering the experience acquired via the use of Tα1 to treat cancer patients, we will also discuss potential approaches for the future introduction of proTα into the clinical setting.

Removal of hepatitis C virus-infected cells by a zymogenized bacterial toxin.

Hepatitis C virus (HCV) infection is a major cause of chronic liver disease and has become a global health threat. No HCV vaccine is currently available and treatment with antiviral therapy is associated with adverse side effects. Moreover, there is no preventive therapy for recurrent hepatitis C post liver transplantation. The NS3 serine protease is necessary for HCV replication and represents a prime target for developing anti HCV therapies. Recently we described a therapeutic approach for eradication of HCV infected cells that is based on protein delivery of two NS3 protease-activatable recombinant toxins we named "zymoxins". These toxins were inactivated by fusion to rationally designed inhibitory peptides via NS3-cleavable linkers. Once delivered to cells where NS3 protease is present, the inhibitory peptide is removed resulting in re-activation of cytotoxic activity. The zymoxins we described suffered from two limitations: they required high levels of protease for activation and had basal activities in the un-activated form that resulted in a narrow potential therapeutic window. Here, we present a solution that overcame the major limitations of the "first generation zymoxins" by converting MazF ribonuclease, the toxic component of the E. coli chromosomal MazEF toxin-antitoxin system, into an NS3-activated zymoxin that is introduced to cells by means of gene delivery. We constructed an expression cassette that encodes for a single polypeptide that incorporates both the toxin and a fragment of its potent natural antidote, MazE, linked via an NS3-cleavable linker. While covalently paired to its inhibitor, the ribonuclease is well tolerated when expressed in naïve, healthy cells. In contrast, activating proteolysis that is induced by even low levels of NS3, results in an eradication of NS3 expressing model cells and HCV infected cells. Zymoxins may thus become a valuable tool in eradicating cells infected by intracellular pathogens that express intracellular proteases.

Gene therapy for pain: results of a phase I clinical trial.

OBJECTIVE: Preclinical evidence indicates that gene transfer to the dorsal root ganglion using replication-defective herpes simplex virus (HSV)-based vectors can reduce pain-related behavior in animal models of pain. This clinical trial was carried out to assess the safety and explore the potential efficacy of this approach in humans. METHODS: We conducted a multicenter, dose-escalation, phase I clinical trial of NP2, a replication-defective HSV-based vector expressing human preproenkephalin (PENK) in subjects with intractable focal pain caused by cancer. NP2 was injected intradermally into the dermatome(s) corresponding to the radicular distribution of pain. The primary outcome was safety. As secondary measures, efficacy of pain relief was assessed using a numeric rating scale (NRS), the Short Form McGill Pain Questionnaire (SF-MPQ), and concurrent opiate usage. RESULTS: Ten subjects with moderate to severe intractable pain despite treatment with >200mg/day of morphine (or equivalent) were enrolled into the study. Treatment was well tolerated with no study agent-related serious adverse events observed at any point in the study. Subjects receiving the low dose of NP2 reported no substantive change in pain. Subjects in the middle- and high-dose cohorts reported pain relief as assessed by NRS and SF-MPQ. INTERPRETATION: Treatment of intractable pain with NP2 was well tolerated. There were no placebo controls in this relatively small study, but the dose-responsive analgesic effects suggest that NP2 may be effective in reducing pain and warrants further clinical investigation.

Cardiac and kidney hormones cure up to 86% of human small-cell lung cancers in mice.

BACKGROUND: Four cardiac hormones synthesized by the same gene, i.e. atrial natriuretic peptide, vessel dilator, long acting natriuretic peptide and kaliuretic peptide, and the kidney hormone urodilatin have anticancer effects in vitro. MATERIALS AND METHODS: These cardiac hormones and urodilatin were infused subcutaneously for 28 days with weekly fresh hormones since they lose biological effects at body temperature for more than a week at 0.3 nm kg(-1) body weight in athymic mice bearing human small-cell lung carcinomas. RESULTS: Long acting natriuretic peptide, vessel dilator, kaliuretic peptide, atrial natriuretic peptide and urodilatin eliminated 86%, 71%, 57%, 43% (P < 0.001 for the cardiac hormones) and 25% (P < 0.05; urodilatin) of the human small-cell lung carcinomas. The treated small-cell lung carcinomas that were not cured grew rapidly, similar to the untreated controls, whose volume was 7 fold larger in 1 week, 18-fold increased in 2 weeks, 39-fold increased in 3 weeks, 63-fold increased in 1 month and 97-fold increased in volume in 6 weeks. One vessel dilator treated small-cell lung carcinoma animal developed a large tumour (8428 mm3 volume) on treatment and this tumour was eliminated with utilizing atrial natriuretic peptide and then long acting natriuretic peptide sequentially. CONCLUSIONS: Four cardiac hormones eliminate up to 86% of human small-cell lung carcinomas in athymic mice. Urodilatin can also eliminate small-cell lung carcinomas but at a lower cure rate of 25%. Unresponsive lesions can be eliminated by utilizing different hormones synthesized by the atrial natriuretic peptide gene in a sequential manner.

Granulin-epithelin precursor as a therapeutic target for hepatocellular carcinoma.

UNLABELLED: Primary liver cancer, hepatocellular carcinoma (HCC), is the fifth most common cancer and the third leading cancer killer in the world. There is no effective therapeutic option for most HCC patients. A new therapeutic strategy is essential. Granulin-epithelin precursor (GEP, also called progranulin, acrogranin, or PC-derived growth factor) was identified as a potential therapeutic target for HCC from our earlier genome-wide expression profiles. We aimed to conduct a detailed investigation with in vitro and animal experiments. We developed the anti-GEP monoclonal antibody (mAb), and examined its effect on hepatoma cells and normal liver cells in vitro. A nude mice model transplanted with human HCC was used to investigate if anti-GEP mAb can inhibit tumor growth in vivo. We demonstrated that anti-GEP mAb inhibited the growth of hepatoma cells but revealed no significant effect on normal liver cells. In the nude mice model transplanted with human HCC, anti-GEP mAb decreased the serum GEP level and inhibited the growth of established tumors in a dose-dependent manner. The anti-GEP mAb reduced tumor cell proliferation via the p44/42 MAPK and Akt pathways, and reduced tumor angiogenesis to deprive the nutrient supply with reduced microvessel density and tumor vascular endothelial growth factor level. CONCLUSION: We have shown that anti-GEP antibody can inhibit HCC growth, providing evidence that GEP is a therapeutic target for HCC treatment.

Effect of prepro-calcitonin gene-related peptide-expressing endothelial progenitor cells on pulmonary hypertension.

BACKGROUND: Calcitonin gene-related peptide (CGRP) is a potent smooth muscle cell proliferation inhibitor and vasodilator. It is now believed that CGRP plays an important role in maintaining a low pulmonary vascular resistance. We evaluated the therapeutic effect of intravenously administered CGRP-expressing endothelial progenitor cells (EPCs) on left-to-right shunt-induced pulmonary hypertension in rats. METHODS: Endothelial progenitor cells were obtained from cultured human peripheral blood mononuclear cells. The genetic sequence for CGRP was subcloned into cultured EPCs by human expression plasmid. Pulmonary hypertension was established in immunodeficient rats with an abdominal aorta to inferior vena cava shunt operation. The transfected EPCs were injected through the left jugular vein at 10 weeks after the shunt operation. Mean pulmonary artery pressure and total pulmonary vascular resistance were detected with right cardiac catheterization at 4 weeks. The distribution of EPCs in the lung tissue was examined with immunofluorescence technique. Histopathologic changes in the structure of the pulmonary arteries was observed with electron microscopy and subjected to computerized image analysis. RESULTS: The lungs of rats transplanted with CGRP-expressing EPCs demonstrated a decrease in both mean pulmonary artery pressure (17.64 +/- 0.79 versus 22.08 +/- 0.95 mm Hg; p = 0.018) and total pulmonary vascular resistance (1.26 +/- 0.07 versus 2.45 +/- 0.18 mm Hg x min/mL; p = 0.037) at 4 weeks. Immunofluorescence revealed that intravenously administered cells were incorporated into the pulmonary vasculature. Pulmonary vascular remodeling was remarkably attenuated with the administration of CGRP-expressing EPCs. CONCLUSIONS: The transplantation of CGRP-expressing EPCs may effectively attenuate established pulmonary hypertension and exert reversal effects on pulmonary vascular remodeling. Our findings suggest that the therapy based on the combination of both CGRP transfection and EPCs may be a potentially useful strategy for the treatment of pulmonary hypertensive disorders.

Leucine at the carboxyl-terminal of endokinins C and D contributes to elicitation of the antagonistic effect on substance P in rat pain processing.

Endokinins are tachykinin peptides designated from a human preprotachykinin C (PPT-C, TAC4) gene and consist of endokinin A (EKA), endokinin B (EKB), endokinin C (EKC) and endokinin D (EKD). A representative of mammalian tachykinins is substance P (SP), which functions as a neurotransmitter or modulator in the pain system; however, little is known about the role of these endokinins, especially EKC and EKD, in pain processing. Therefore, we evaluated the effects of EKC/D (using the common carboxyl-terminal duodecapeptide in EKC and EKD) on pain processing in rats. Pretreatment with EKC/D prevented induction of scratching behavior and thermal hyperalgesia by intrathecal administration of EKA/B (using the common C-terminal decapeptide in EKA and EKB) and SP and c-Fos expression in laminae I/II and V/VI of the spinal cord by noxious thermal stimulation. A prominent difference between EKC/D and SP is the presence of leucine instead of methionine at the carboxyl-terminal of EKC/D. Thus, to clarify whether leucine at the carboxyl-terminal of EKC/D plays an important role in determining the inhibitory effect of this peptide, we intrathecally administered [Met(12)]-EKC/D in which only leucine of EKC/D is replaced by methionine. This peptide did not exhibit the inhibitory effect on SP-induced scratching behavior or thermal hyperalgesia but conversely caused thermal hyperalgesia. Taken together, these findings indicate that EKC/D has an inhibitory effect on pain processing in the rat spinal cord, and the effect is due to leucine at the carboxyl-terminal of EKC/D.