Inhibitor Diprotin Research Articles

The DPP4 Dependent Transcriptome Identifies Pathways to Improve Mouse Hematopoietic Cell Function

Hematopoietic cell transplantation (HCT) is a curative treatment for hematologic disorders wherein healthy donor hematopoietic stem (HSCs) and progenitor cells (HPCs) collected from bone marrow (BM), mobilized peripheral blood (PB) or umbilical cord blood (CB) reconstitute an ablated hematopoietic system. There is a strong need to enhance functional competency of HSCs/HPCs for HCT to prevent complications like graft versus host disease (GVHD), relapse, and graft failure. Dipeptidyl peptidase 4 (DPP4) is a protein that cleaves N-terminal dipeptides from hematopoietic factors, impacting their function. Inhibition of DPP4 improves HSC/HPC competency in mouse models of HCT and clinically speeds neutrophil recovery after CB transplantation and reduces incidences of GVHD after PB transplantation. Thus, DPP4 inhibition is a viable component of a multi-pronged strategy to improve engraftment and prevent GVHD. While DPP4 inhibition improved early neutrophil recovery in clinical trials, the effects were modest, required high dose treatment, and effects on platelet recovery were unclear. Examining mechanisms by which DPP4 inhibition drives HSC/HPC functional competency may yield insight into targets that can enhance DPP4 driven improvements to HCT. We harvested BM from mice treated with the DPP4 inhibitor Diprotin A (DPA) and sorted immunophenotypically defined HSCs, multipotent progenitors (MPPs), common myeloid progenitors (CMPs), and granulocyte-macrophage progenitors (GMPs) for transcriptomic analysis. Linear modeling to examine differential gene expression controlling for cell type revealed that DPP4 inhibition gave strong enrichment of gene programs associated with mitochondrial function. Using a mitochondrial stress test and the Seahorse metabolic flux analyzer, we found that lineage depleted mouse BM cells treated ex vivo with DPA had lower basal oxygen consumption rates and spare respiratory capacity. These seemingly paradoxical data are consistent with reports that highly functional HSCs have higher mitochondrial mass but exhibit less mitochondrial activity. Thus, DPP4 inhibition may preserve high levels of functional competency by reducing cellular dependence on oxidative phosphorylation. To examine DPP4 inhibition effects on individual subpopulations of HSCs/HPCs, we modeled each cell type separately for differential gene expression. We found that gene programs associated with neutrophil development were significantly enriched in HSCs and MPPs from mice treated with DPA, including genes Elane, Lcn2, Prtn3 and Ctsg/h, concomitant with downregulation of platelet development programs in HSCs and GMPs from mice treated with DPA, including genes Gp6/9, P2ry12 and Clec1b. This suggests that DPP4 drives improved engraftment in part by priming HSCs/HPCs for neutrophil recovery but does not enhance platelet development. We then sought to target a platelet activating pathway in combination with DPP4 inhibition to improve HCT. Recipient mice were treated on days -2, -1, and 0 with 75 mg/kg DPA and day 0 with 50 µg/kg and days 1, 3, 5, and 7 with 25 µg/kg recombinant human Thrombopoietin (TPO), a platelet activating agonist. On day -1 mice were lethally irradiated and transplanted via tail vein with 2.5x10 5 whole BM donor cells on day 0. Complete blood counts 2 weeks following transplantation showed that mice treated with DPA alone exhibited worse platelet recovery, in line with our transcriptomic data. However, mice treated with the combination of DPA and TPO exhibited significantly better platelet recovery than vehicle controls and DPA alone. Further, neutrophil recovery trended towards improved recovery in the combination treatment compared to the single agent treatments. Flow cytometry of the PB revealed no differences in BM homing or in donor PB chimerism one month following HCT in the combination treatment compared to single agents (though all treatments showed improved homing over vehicle controls), suggesting the primary effects were on early recovery and not on initial or long-term engraftment. These data elucidate the DPP4 dependent transcriptome in the hematopoietic compartment, providing molecular insights into genes that drive enhanced functional competency of HSCs/HPCs. We also demonstrate that the DPP4 dependent transcriptome can reveal gene programs that can be targeted in combination to provide additive improvement on transplantation recovery.

Anti-hyperglycemic Δ5 steroids, marginoids A-C from marine veined octopus Amphioctopus marginatus (Octopodidae): Prospective natural leads inhibit serine exopeptidase dipeptidyl peptidase-4

Three Δ5 steroid analogues, marginoids A-C were purified from the organic extract of marine veined octopus Amphioctopus marginatus (Taki, 1964) (family Octopodidae) distributed on the Asian and Mediterranean coasts. Their structures were elucidated as (5Z)-3β-acetoxy-cholesta-5-en-25-ethylene-22β-hydroxy-23,26-lactone (marginoid A), (5Z, 25Z)-3β-yl-(1′-(E)-3′-hydroxy-4′-methyl-hex-5′-enoate)-22-oxo-26-furanyl-cholesta-5,25-diene (marginoid B), and (5Z)-3β-yl-(7′-methoxypropan-8′-yl)-tetrahydro-2H-pyran-2-one-cholesta-5,24-dien (marginoid C) based on extensive spectroscopic experiments. Marginoid B with hydroxyl-methyl-hexanoate at the C-3 position in conjunction with the heterocyclic furanyl ring displayed superior anti-hyperglycemic properties as acknowledged by its promising serine protease dipeptidyl peptidase-4 attenuation potential (IC50 3.49 µM) displaying comparable activity with the standard DPP-4 inhibitor (DPP-4i) diprotin A (IC50 4.53 µM). The anti-hyperglycemic properties were corroborated by the promising antioxidant activities (IC50 ∼ 0.8–1.0 mM) of these Δ5 steroids, marginoids A-C. Sizeably greater electronic properties, balanced hydrophobic-lipophilic properties (log POW 6.4–8.3), and comparatively lower steric factors were directly proportional to their bioactive properties. Molecular simulation studies in the binding sites of DPP-4 and lesser binding energy (-12.17 kcal/mol) and inhibition constant (Ki 1.20 nM) of marginoid B could be correlated with anti-hyperglycemic properties. Promising bioactivities of marginoid B isolated from A. marginatus are anticipated for nutraceutical applications against hyperglycemia.

Diprotin A TFA Exerts Neurovascular Protection in Ischemic Cerebral Stroke.

BackgroundIt has been established that the dipeptidyl peptidase-4 (DPP-4) inhibitor Diprotin A TFA can reduce vascular endothelial (VE)-cadherin disruption by inhibiting the increase in cleaved β-catenin in response to hypoxia, thereby protecting the vascular barrier of human umbilical vein endothelial cells. In this study, we sought to investigate the possible effect of Diprotin A TFA on the VE barrier after cerebral ischemic stroke in mice.MethodsC57BL/6J mice were divided into five groups, namely, (1) sham, (2) stroke, (3) stroke + dimethyl sulfoxide (DMSO), (4) stroke + Diprotin A TFA, and (5) stroke + Diprotin A TFA + XAV-939. First, the cerebral ischemia model was established by photothrombotic ischemia, followed by intraperitoneal injection with Diprotin A TFA and XAV-939 at doses of 70 μg/kg and 40 mg/kg 30 min once in the morning and once in the evening for 3 days. Immunofluorescence staining and Western blot methods were used to analyze the expression of vascular and blood-brain barrier (BBB)-associated molecular markers in the peri-infarct area.ResultsCompared with the vehicle control group, we found that mice injected with Diprotin A TFA exhibited reduced cerebral infarction volume, increased vascular area and length around the brain injury, increased pericyte and basement membrane coverage, upregulated expression of BBB tight junction proteins, and improved their BBB permeability, whereas the group injected with both drug and inhibitor exhibited significantly aggravated vascular injury and BBB permeability.ConclusionDiprotin A TFA can reduce VE-cadherin disruption by inhibiting ischemia-hypoxia-induced β-catenin cleavage to protect blood vessels.

Rapid Isolation of a Dipeptidyl Peptidase IV Inhibitor from Fritillaria cirrhosa by Thin-Layer Chromatography–Bioautography and Mass Spectrometry-Directed Autopurification System

A thin-layer chromatography (TLC)-bioautography-mass spectrometry (MS) method coupled with a liquid chromatography- MS-controlled autopurification system was developed and applied for screening and isolating natural dipeptidyl peptidase IV (DPP IV) inhibitor from plant extracts. An unknown constituent with potential DPP IV inhibitory activity from bulbs of Fritillaria cirrhosa was discovered using TLC-bioautography, followed by using a TLC interface with mass spectrometry to obtain m/z of the target compound, and the purification of the compound was directly achieved with a mass-directed autopurification system for 40 runs of injection. Finally, 2.1 mg of the compound was obtained and identified as peimisine by 1H and 13C nuclear magnetic resonance (NMR) spectroscopy. The half maximal inhibitory concentration (IC50) of DPP IV inhibitory activity was determined at 80.5 μm comparing to 58.0 μm of the standard DPP IV inhibitor diprotin A by a spectrophotometric method.

Inhibiting DPP4 in a mouse model of HHT1 results in a shift towards regenerative macrophages and reduces fibrosis after myocardial infarction.

AimsHereditary Hemorrhagic Telangiectasia type-1 (HHT1) is a genetic vascular disorder caused by haploinsufficiency of the TGFβ co-receptor endoglin. Dysfunctional homing of HHT1 mononuclear cells (MNCs) towards the infarcted myocardium hampers cardiac recovery. HHT1-MNCs have elevated expression of dipeptidyl peptidase-4 (DPP4/CD26), which inhibits recruitment of CXCR4-expressing MNCs by inactivation of stromal cell-derived factor 1 (SDF1). We hypothesize that inhibiting DPP4 will restore homing of HHT1-MNCs to the infarcted heart and improve cardiac recovery.Methods and resultsAfter inducing myocardial infarction (MI), wild type (WT) and endoglin heterozygous (Eng+/-) mice were treated for 5 days with the DPP4 inhibitor Diprotin A (DipA). DipA increased the number of CXCR4+ MNCs residing in the infarcted Eng+/- hearts (Eng+/- 73.17±12.67 vs. Eng+/- treated 157.00±11.61, P = 0.0003) and significantly reduced infarct size (Eng+/- 46.60±9.33% vs. Eng+/- treated 27.02±3.04%, P = 0.03). Echocardiography demonstrated that DipA treatment slightly deteriorated heart function in Eng+/- mice. An increased number of capillaries (Eng+/- 61.63±1.43 vs. Eng+/- treated 74.30±1.74, P = 0.001) were detected in the infarct border zone whereas the number of arteries was reduced (Eng+/- 11.88±0.63 vs. Eng+/- treated 6.38±0.97, P = 0.003). Interestingly, while less M2 regenerative macrophages were present in Eng+/- hearts prior to DipA treatment, (WT 29.88±1.52% vs. Eng+/- 12.34±1.64%, P<0.0001), DPP4 inhibition restored the number of M2 macrophages to wild type levels.ConclusionsIn this study, we demonstrate that systemic DPP4 inhibition restores the impaired MNC homing in Eng+/- animals post-MI, and enhances cardiac repair, which might be explained by restoring the balance between the inflammatory and regenerative macrophages present in the heart.

Molecular cloning and biochemical characterization of Xaa-Pro dipeptidyl-peptidase from Streptococcus mutans and its inhibition by anti-human DPP IV drugs.

Streptococcus mutans harbours an intracellular, human DPP IV-analogous enzyme Xaa-Pro dipeptidyl-peptidase (EC 3.4.14.11). According to previous reports, an extracellular isozyme in S. gordonii and S. suis has been associated with virulence. Speculating that even an intracellular form may aid in virulence of S. mutans, we have tried to purify, characterize and evaluate enzyme inhibition by specific inhibitors. The native enzyme was partially purified by ion-exchange and gel filtration chromatography. Owing to low yield, the enzyme was overexpressed in Lactococcus lactis and purified by affinity chromatography. The recombinant enzyme (rSm-XPDAP) had a specific activity of 1070 U mg(-1), while the Vmax and Km were 7 μM min(-1) and 89 ± 7 μM (n = 3), respectively. The serine protease inhibitor phenylmethylsulphonyl fluoride and a DPP IV-specific inhibitor diprotin A proved to be active against rSm-XPDAP. As a novel approach, the evaluation of the effect of anti-human DPP IV (AHD) drugs on rSm-XPDAP activity found saxagliptin to be effective to some extent (Ki = 129 ± 16 μM), which may lead to the synthesis and development of a new class of antimicrobial agents.

Effect of Diprotin A, an Inhibitor of Dipeptidyl Peptidase IV, on Immunological Parameters of Lymphocytes in Intact Animals and Animals with Experimental Autoimmune Process.

We found the peculiarities of the effects of dipeptidyl peptidase IV inhibitor diprotin A on immunological parameters of lymphocytes in lymphoid organs in intact animals and in animals with experimental autoimmune process. In intact animals, diprotin A increased cellularity of the thymus and spleen, number of CD4+, CD8+, and CD4+8+ thymocytes and CD3+ splenocytes and reduced cellularity of lymph nodes and the number of antibody-forming cells in the spleen. Diprotin A administered against the background of autoimmune process, reduced thymus cellularity, number of antibody-forming cells in the spleen, and number of CD4 and CD4+8+ thymocytes in comparison with the corresponding parameters of autoimmune process.

Dipeptidyl peptidase IV reduces trophoblast invasion by inhibiting the activity of MMPs.

Preeclampsia is a severe pregnancy complication in part due to insufficient implantation. This study aimed at elucidating the mechanism of action of dipeptidyl peptidase IV (DPPIV) in preeclampsia. Small activating RNAs (saRNA) were used to upregulate DPPIV expression in human trophoblast JAR cells. The DPPIV expression level was analyzed by real-time quantitative PCR and western blot and its activity was measured by luminescent protease assay. MMP-9 activity was analyzed by zymography and cell invasion by matrigel invasion assay. DPPIV expression level and activity was significantly increased by saRNA in JAR cells. DDPIV specific inhibitor diprotin A inhibited its activity at both basal and activated levels. DPPIV did not regulate MMP-9 expression but did repress MMP-9 activity. The invading ability of JAR cells was reduced by saRNA but increased by diprotin A. DPPIV might be responsible for the shallow implantation of the placenta due to its inhibition of the invading ability of extravillous trophoblasts, causing preeclampsia at later stage of pregnancy.

Sinusoidal Endothelial CD26 Cleavage of Neuropeptide Y Regulates Transendothelial Migration and Mobilization of Hematopoietic Stem and Progenitor Cells in Response to G-CSF.

Abstract 2347Hematopoietic stem and progenitor cell (HSPC) mobilization by G-CSF is reduced in CD26 knockout (−/−) mice or mice treated with compounds that inhibit CD26 enzyme activity. However, how CD26 regulates mobilization is not known. CD26 is a surface dipeptidyl peptidase expressed on hematopoietic and non-hematopoietic cells. The fact that CD26 is expressed on HSPC, has suggested that intrinsic HSPC CD26 is involved in their mobilization. We reasoned that if intrinsic HSPC CD26 is crucial for their mobilization, then CD26+ HSPC would mobilize more readily than CD26− HSPC. After G-CSF treatment, CD26+ SLAM LSK and LSK cells increased 2 and 3.7-fold in peripheral blood (PB) whereas CD26− SLAM LSK and LSK cells increased 3.9 and 8.7 fold respectively; with the ratio of CD26+/CD26− HSPC in the bone marrow (BM) remaining unchanged. In chimeric mice where hematopoietic cells were either wild-type or CD26−/− in origin and the stromal environment was wild-type, equivalent CFU-GM and LSK cells were mobilized by G-CSF. These studies suggest that CD26 expression on hematopoietic origin cells including HSPC is not required for the G-CSF induced mobilization of HSPC.The BM stromal environment includes both osteolineage cells and endothelial cells (EC). To explore whether osteolineage cell CD26 is involved in G-CSF mobilization, we quantitated CD45−CD119−CD31−Sca-1+ Alcam− cells enriched for mesenchymal progenitor cells (MPC) and CD45−CD119−CD31− Sca-1- Alcam+ cells enriched for osteoblasts (OB) in the BM of mice treated with G-CSF or G-CSF plus the CD26 inhibitor diprotin A. Total MPC and OB number as well as the retention factors SDF-1, SCF-1 and osteoprotegerin (OPG) were reduced equally in the BM of G-CSF and G-CSF plus diprotin A treated mice, suggesting that osteolineage cell CD26 is not involved in mobilization. Since endothelial cells (EC) express CD26 and HSPC resides proximal to sinusoidal endothelial cells (SEC), we evaluated if SEC CD26 has a role in G-CSF induced HSPC mobilization. G-CSF treatment increased CD26 expression on CD45- Ter119− Sca-1− VEGFR3+ SEC by 2-fold. Because mobilized HSPC must transmigrate across SEC to enter the PB, we determined if EC CD26 regulates HSPC transmigration. Treatment of confluent EC monolayers with G-CSF in vitro, increased transendothelial migration of both wild-type and CD26−/−HSPC, which was significantly reduced by diprotin A, supporting the hypothesis that enhanced expression of CD26 on BM SEC by G-CSF is involved in HSPC mobilization.Since SDF-1 is a BM retention factor for HSPC and CD26 can cleave SDF-1 in vitro, it is possible that SDF-1 degradation by CD26 plays a role in HSPC egress. However, direct evidence supporting proteolytic inactivation of SDF-1 by CD26 in response to G-CSF is not available. Using a novel bio-binding assay and mass spectroscopy, we found similar levels of active SDF-1 in the BM extracellular fluid and on SEC membranes from G-CSF treated wild-type and CD26−/− mice, suggesting that G-CSF mobilization is not linked to SDF-1degradation. We therefore searched protein sequence databases for known EC products containing a CD26 recognition site and implicated in HSPC trafficking. We found that the neurotransmitter neuropeptide Y (NPY), a sympathetic nerve and EC product contains an N-terminal Tyr-Pro sequence and can be cleaved to NPY3–36 by CD26. We hypothesized that if cleavage of NPY by EC CD26 is involved in transendothelial migration of HSPC, then reduced HSPC migration through EC monolayers in the presence of G-CSF plus diprotin A would be reversed in the presence of NPY3–36. As expected, NPY3–36 reversed the diprotin A mediated attenuation in HSPC migration through G-CSF treated EC. In vivo, the attenuated response to G-CSF mobilization in diprotin A treated or CD26−/− mice could be completely reversed by co-administration of NPY3–36, but not full length NPY. In G-CSF mobilized wild-type mice, we found that expression of gap junction proteins on BM SEC, including CD31 and VE-cadherin are reduced coincident with increased EC permeability, which is attenuated by co-administration of diptotin A and restored to normal in mice receiving G-CSF plus diprotin A and NPY3–36, but not full length NPY. In conclusion, we have found that CD26 regulates G-CSF induced mobilization that is independent of the SDF-1 paradigm; rather elevated SEC CD26 in response to G-CSF enhances transendothelial migration and mobilization of HSPC via cleavage of NPY. Disclosures:No relevant conflicts of interest to declare.

Loss of CD26 protease activity in recipient mice during hematopoietic stem cell transplantation results in improved transplant efficiency

A firm understanding of the biology of hematopoietic stem and progenitor cell (HSC/HPC) trafficking is critical to improve transplant efficiency and immune reconstitution during hematopoietic stem cell transplantation (HSCT). Our earlier findings suggested that suppression of CD26 (dipeptidyl peptidase IV) proteolytic activity in the donor cell population can be utilized as a method for increasing transplant efficiency. However, factors in the recipient should not be overlooked, given the potential for the bone marrow (BM) microenvironment to regulate HSCT. We first evaluated CD26 expression and then investigated the effects of the CD26 inhibitor diprotin A and the absence of CD26 (CD26-/-) in recipient mice on HSC/HPC homing and engraftment using an in vivo congenic mouse model of HSCT. A significant increase in donor cell engraftment into the peripheral blood (PB), and to a lesser extent homing into the BM, was observed in CD26-/- mice or CD26 inhibitor-treated mice. Increased PB engraftment of CD26-/- mice was significant at 3 and 6 months, but not 1 month, after transplant. It was noted that the increased homing was statistically greater with donor cell manipulation (CD26-/- donor cells) than with recipient manipulation (CD26-/- recipient mice). Conversely, donor and recipient manipulation both worked well to increase PB engraftment at 6 months. These results provide preclinical evidence of CD26, in the HSCT recipient, as a major regulator of HSC/HPC engraftment with minor effects on HSC/HPC homing and suggest the potential use of CD26 inhibitors in HSCT patients to improve transplant efficiency.

CD26 protease inhibition improves functional response of unfractionated cord blood, bone marrow, and mobilized peripheral blood cells to CXCL12/SDF-1

Hematopoietic stem cell transplantation (HSCT) is an important treatment option for patients with malignant and nonmalignant hematologic diseases. Methods to improve transplant efficiency are being explored with the intent to improve engraftment and immune reconstitution post-HSCT. A current approach under investigation involves treatment of donor cells with inhibitors that target the protease CD26, a negative regulator of the chemokine CXCL12/stromal cell-derived factor-1. CD26 inhibitor treatment has been shown to improve the functional response of CD34(+) cord blood (CB) cells, but not CD34(+) granulocyte colony-stimulating factor-mobilized peripheral blood stem cells, to CXCL12/stromal cell-derived factor-1. The effect of CD26 inhibitors on unfractionated CB, bone marrow, or granulocyte colony-stimulating factor-mobilized peripheral blood mononuclear cells has not been evaluated previously. We observed that although CB had greater CD26 expression than bone marrow or mobilized peripheral blood, treatment with a CD26 inhibitor (Diprotin A) resulted in increased responsiveness to stromal cell-derived factor-1 for all three mononuclear cell sources tested. This suggests that clinical therapeutic benefit might be gained by using CD26 inhibitors as a strategy to improve engraftment of unfractionated mobilized peripheral blood cells as well as CB cells.

Influence of estradiol supplementation on neuropeptide Y neurotransmission in skeletal muscle arterioles of F344 rats

The effects of estradiol on neuropeptide Y (NPY) neurotransmission in skeletal muscle resistance vessels have not been described. The purpose of this study was to determine the effects of long-term estradiol supplementation on NPY overflow, degradation, and vasoconstriction in gastrocnemius first-order arterioles of adult female rats. Female rats (4 mo; n = 34) were ovariectomized (OVX) with a subset (n = 17) receiving an estradiol pellet (OVE; 17β-estradiol, 4 μg/day). After conclusion of the treatment phase (8 wk), arterioles were excised, placed in a physiological saline solution (PSS) bath, and cannulated with micropipettes connected to albumin reservoirs. NPY-mediated vasoconstriction via a Y(1)-agonist [Leu31Pro34]NPY decreased vessel diameter 44.54 ± 3.95% compared with baseline; however, there were no group differences in EC(50) (OVE: -8.75 ± 0.18; OVX: -8.63 ± 0.10 log M [Leu31Pro34]NPY) or slope (OVE: -1.11 ± 0.25; OVX: -1.65 ± 0.34% baseline/log M [Leu31Pro34]NPY). NPY did not potentiate norepinephrine-mediated vasoconstriction. NPY overflow experienced a slight increase following field stimulation and significantly increased (P < 0.05) over control conditions in the presence of a DPPIV inhibitor (diprotin A). Estradiol status did not affect DPPIV activity. These data suggest that NPY can induce a moderate decrease in vessel diameter in skeletal muscle first-order arterioles, and DPPIV is active in mitigating NPY overflow in young adult female rats. Long-term estradiol supplementation did not influence NPY vasoconstriction, overflow, or its enzymatic breakdown in skeletal muscle first-order arterioles.

Granulocyte colony-stimulating factor treatment plus dipeptidylpeptidase-IV inhibition augments myocardial regeneration in mice expressing cyclin D2 in adult cardiomyocytes

Although pharmacological interventions that mobilize stem cells and enhance their homing to damaged tissue can limit adverse post-myocardial infarction (MI) remodelling, cardiomyocyte renewal with this approach is limited. While experimental cell cycle induction can promote cardiomyocyte renewal following MI, this process must compete with the more rapid processes of scar formation and adverse remodelling. The current study tested the hypothesis that the combination of enhanced stem cell mobilization/homing and cardiomyocyte cell cycle induction would result in increased myocardial renewal in injured hearts. Myocardial infarction was induced by coronary artery ligation in adult MHC-cycD2 transgenic mice (which exhibit constitutive cardiomyocyte cell cycle activity) and their non-transgenic littermates. Mice were then treated with saline or with granulocyte colony-stimulating factor (G-CSF) plus the dipeptidylpeptidase-IV (DPP-IV) inhibitor Diprotin A (DipA) for 7 days. Infarct thickness and cardiomyocyte number/infarct/section were significantly improved in MHC-cycD2 mice with G-CSF plus DipA treatment when compared with MHC-cycD2 transgene expression or G-CSF plus DipA treatment alone. Echocardiographic analyses revealed that stem cell mobilization/homing and cardiomyocyte cell cycle activation had an additive effect on functional recovery. These data strongly suggest that G-CSF plus DPP-IV inhibition, combined with cardiomyocyte cell cycle activation, leads to enhanced myocardial regeneration following MI. The data are also consistent with the notion that altering adverse post-injury remodelling renders the myocardium more permissive for cardiomyocyte repopulation.

CD26 Inhibition Preferentially Enhances In Vitro Migration of G-CSF + Plerixafor (AMD3100) Mobilized PB as Compared to G-CSF Mobilized PB In Multiple Myeloma Autografts.

AbstractAbstract 3714Hematopoietic stem and progenitor cell (HSC/HPC) trafficking into the bone marrow during hematopoietic stem cell transplantation (HSCT) is thought to be an important biological step that can be targeted to improve transplant efficiency and subsequent immune reconstitution. It has been reported that loss/inhibition of the peptidase CD26, also known as DPPIV (dipeptidylpeptidase IV), increases the transplant efficiency of mouse HSC/HPC (Christopherson, KW 2nd, et al. Science 2004). In a similar manner, inhibition of CD26 on CD34+ or lin− cord blood (CB) cells improves in vitro cell migration and in vivo engraftment into immunodeficient mice (Christopherson, KW 2nd, et al. Stem Cells and Dev 2007). There is currently controversy as to whether inhibition of CD26 on mobilized peripheral blood (mobPB) has benefit since it has been reported that inhibition of CD26 does not improve migration of G-CSF mobPB CD34+ cells in response to SDF-1 (Kawai K, et al. Stem Cells and Dev 2007. 16:361–70) but does improve the migration of G-CSF mobPB mononuclear cells (MNC) (Frank R, et al. ASH Abstract #372, 2009). Additionally, it has been reported that AMD3100 mobPB c-kit+ mouse cells have high CD26 expression similar to BM in contrast to G-CSF mobPB c-kit+ mouse cells which have very little CD26 expression (Bonig H, Exp Hem, 2009). The combination G-CSF + AMD3100 (a CXCR4 antagonist also known as plerixafor or Mozobil ®) has been recently introduced into the clinic for use in obtaining mobPB from Multiple Myeloma patients. We therefore investigated whether CD26 on human mobPB MNC obtained from Multiple Myeloma autografts mobilized with either G-CSF or G-CSF + plerixafor would have an effect on SDF-1 induced cell migration. To do this, non-adherent MNC from mobPB were obtained from discarded bags following the completion of stem cell infusion of previously frozen autografts. CD26 expression was detected by flow cytometery. CD26 activity was determined using the substrate Gly-Pro P-nitroanilide (Gly-Pro-pNA). To assess migration, chemotaxis assays were performed by loading 5×104 cells into the upper chamber of a 96 well transwell plate, adding human SDF-1α (0–400 ng/mL) in the lower chamber, and incubating for 2 hours at 37°C, 5% CO2. By flow cytometry, CD26 expression was 21.61±2.19% and 21.65±3.68% of the CD45+ cell population for G-CSF and G-CSF + plerixafor mobPB MNCs respectively. CD26 activity was 15.4±1.2 pmol pNA/minute and 16.3±0.4 pmol pNA/minute for G-CSF and G-CSF + AMD3100 MNCs. During functional in vitro assays, cells were cultured with or without a 5mM concentration of the CD26 inhibitor Diprotin A (DpA). DpA treatment of G-CSF + plerixafor mobPB resulted in a significant increase in cell migration as compared to untreated cells. Specifically, the percent migration at 400ng/mL SDF-1α was 21.3±2.4% and 31.6±3.0% for untreated and DpA treated G-CSF + plerixafor mobPB cells respectively (p=<0.01, n=10). The percent migration in response to 400ng/mL SDF-1α was 19.8±2.0% and 25.3±2.6% for untreated and DpA treated G-CSF mobPB cells respectively (p=0.21, n=7). These data indicate that CD26 inhibition, by treatment with DpA, results in a preferential increase in SDF-1 induced migration of G-CSF + plerixafor mobPB cells as compared to G-CSF mobPB cells. The preferential response does not appear to correlate to any change in CD26 expression or activity since both types of mobPB have equivalent expression and activity prior to the migration assay. Our data suggest that un-fractionated mobPB collected following mobilization using the combination of G-CSF + plerixafor (AMD3100) may have inherently different cell trafficking regulatory properties when compared to mobPB collected following G-CSF alone and that additional regulatory mechanisms allow the G-CSF + plerixafor mobPB to have an enhanced response to SDF-1 in the absence of CD26 suppression. Additionally, our data suggest that inhibition of CD26 peptidase activity may be more clinically relevant in the G-CSF + plerixafor mobilized patient population with regard to achieving improved stem cell trafficking. Disclosures:Fung:Genzyme: Honoraria, Speakers Bureau.

Cytokine Treatment of CD34+ Cord Blood Cells with G-CSF, GM-CSF, or SCF During 48 Hours of Ex Vivo Culture Alters CD26/DPPIV Peptidase Activity and Subsequent Engraftment Into NSG Immunodeficient Mice.

AbstractAbstract 1460Many patients with hematologic malignancies choose hematopoietic stem cell transplantation (HSCT) as a treatment option. The most common source of Hematopoietic Stem and Progenitor Cells (HSC/HPC) for adult recipients is mobilized Peripheral Blood (mobPB). Limited quantities of HSC/HPC obtainable from an umbilical cord restricts its use for adult recipients. Ex vivo treatment of umbilical cord blood (CB) with cytokines and growth factors is being used to expand the population of cord blood HSC/HPCs in hopes of obtaining higher numbers of transplantable CB cells. In addition, cytokines and growth factors are often utilized post-transplant in an attempt to improve the rate of immune reconstitution. It has been previously reported that granulocyte-colony-stimulating factor (G-CSF), and granulocyte-macrophage-colony-stimulating factor (GM-CSF) up-regulate CD26 (dipeptidyl peptidase IV/DPPIV) activity on freshly isolated CD34+ CB cells within 18 hours of culture [Christopherson, et al. Exp Hematol 2006]. Separate studies have demonstrated that treatment of uncultured CD34+ CB cells with the CD26 inhibitor Diprotin A increases transplant efficiency into immunodeficient mice [Christopherson, et al. Stem Cells Dev. 2007]. We evaluated here the in vitro and in vivo effects of CD26 inhibitor treatment on previously frozen CB CD34+ cells cultured ex vivo with G-CSF, GM-CSF or SCF for 48 hours. We examined CD26 expression by multivariate flow cytometry, CD26 activity using the established chromogenic CD26 substrate, Gly-Pro-p-nitroanilide (Gly-Pro-pNA), and SDF-1α induced migration and adhesion. In vivo, we examined long-term engraftment in NSG (NOD/SCID/IL2Rγnull) immunodeficient mice. After 48 hours of culture with cytokine treatment we observed altered CD26 expression on CD34+ CB cells. There was both an increase in the percentage of CD26+ cells and the mean fluorescence intensity (MFI) of CD26. Additionally, CD26 activity was 1.20, 1.59, 1.58, and 1.65 fold greater after ex vivo culture in untreated, G-CSF, GM-CSF and SCF treated CB CD34+ cells respectively compared to the CD26 activity prior to culture. The increase in CD26 activity as a result of treatment with G-CSF (p≤ 0.01), GM-CSF (p≤ 0.05) or SCF (p≤ 0.01) was significantly higher than the CD26 activity measured in the untreated cells following 48 hours of culture. Post-culture treatment with the CD26 inhibitor, Diprotin A, significantly improved SDF-1α induced migration and adhesion of cultured CD34+ CB cells in vitro, particularly in G-CSF treated cells (p≤ 0.05). Diprotin A treatment of CD34+ CB cells previously treated with G-CSF also significantly increased the long-term in vivo engraftment of stem and progenitor (CD34+CD38-, p=0.032), monocyte (CD14+, p=0.015), and megakaryocyte/platelet (CD61+, p=0.020) cells in the bone marrow of NSG mice. CD26 has been previously shown to cleave SDF-1 (stromal cell-derived factor 1/CXCL12). After cleavage, SDF-1 retains its ability to bind to its receptor (CXCR4) but no longer signals. SDF-1 is a powerful chemoattractant and has been shown to be important in mobilization, homing, and engraftment of HSCs and HPCs. This study demonstrates the influence of ex vivo culture and the effect of cytokine treatment on CD26 activity and subsequent biologic function related to HSCT. All three cytokines studied caused a significant increase in enzymatic activity at 48 hours compared to untreated cells. The up-regulation of CD26 protein expression caused by cytokine treatment for 48 hours, in particular G-CSF, had a significant impact on SDF-1 stimulated migration and adhesion. This was demonstrated in vitro by the improvement in cell function after CD26 inhibitor treatment and in vivo by the improved engraftment seen in the G-CSF treated cells with CD26 inhibitor treatment. These experiments suggest that combining CD26 inhibitor treatment following culture with G-CSF treatment during culture has the greatest overall benefit in engraftment outcome. By increasing our understanding of the effects of exogenous cytokines during culture on trafficking, ex vivo expanded CB has the potential to become a more effective means of not only increasing numbers of CB HSC/HPCs but also engraftment outcomes. This would ultimately allow expanded cord blood to become a more viable option for HSCT. Disclosures:No relevant conflicts of interest to declare.

W1785 Loss of NLRP3 Signaling Results in Altered Dendritic Cells (DC) Population and Impaired Induction of Regulatory T Cells (Treg): Implications for Intestinal Inflammation

Background: We recently reported that proper blood processing using the RAPID (reduced temperatures, acidification, protease inhibition, isotopic exogenous controls, dilution) method is of key importance before measuring plasma concentration and molecular forms of labile hormones such as acyl ghrelin, cholecystokinin-58, gastrin-releasing peptide and somatostatin-28 (Endocrinology 150:5113-8; 2009). Active glucagon-like peptide-1 (GLP1(7-36)amide and GLP-1(7-37)) can be produced in blood ex-vivo by an endopeptidase cleaving GLP-1(1-36) and GLP-1(1-37) or destroyed by the action of dipeptidyl aminopeptidase IV (DPP-IV) to produce GLP-1(9-36)amide or GLP-1(9-37). Aim: To investigate whether different blood processing influences the detection of active and total GLP-1 plasma levels. Methods: Blood was obtained between 8-9 AM from male Sprague-Dawley rats (300-350g) either fed ad libitum or 24 h fasted (n=5-6/group) and processed in three ways: 1. collected in ice cold tubes containing EDTA (7.5%, 10μl/1ml) followed by centrifugation, 2. same as 1. plus DPP-IV inhibitor (diprotin A, 50μM) or 3. RAPID method without isotopic standards (RAPD). Samples were stored at -80°C until radioimmunoassays for total and active GLP1. Results: Active GLP-1 levels did not change among metabolic conditions irrespective of the blood processing (P 0.05). Plasma formation and DPP-IV inhibition gave comparable results for circulating total GLP-1 plasma levels of ad libitum fed rats while RAPD processing resulted in lower levels (P 0.05). In a preliminary experiment ~50% of iodinated GLP-1(7-36) was lost in Falcon tubes as used in the RAPD method. Conclusion: Active GLP-1 does not change under the metabolic conditions tested. The two plasma formation methods yield similar results for the measurement of circulating active and total GLP-1, whereas RAPD processing unexpectedly results in lower plasma levels of total and active GLP-1. The use of isotopic exogenous standards will be important to determine recovery and peptide structure alteration ex-vivo possibly leading to changed blood levels of active or total GLP-1. NIDDK 41301/70851/73152

Use of the CD26 Inhibitor Diprotin A (Ile-Pro-Ile) to Increase Mobilized Peripheral Blood Mononuclear Cell Adhesion and Migration.

Abstract 372▪▪This icon denotes an abstract that is clinically relevant.A firm understanding of the biology of hematopoietic stem and progenitor cell (HSC/HPC) trafficking is believed to be critical for the development of methodologies to improve transplant efficiency and subsequent immune reconstitution during hematopoietic stem cell transplantation (HSCT) in the clinical setting. We have previously reported that suppression of the peptidase CD26/DPPIV (dipeptidylpeptidase IV) on mouse HSC/HPC as a method of increasing transplant efficiency (Christopherson, KW 2nd, et al. Science 2004. 305:1000-3). We have also described that inhibition of CD26 on CD34+ or lin- cord blood (CB) cells improves engraftment into immunodeficient mice (Christopherson, KW 2nd, et al. Stem Cells and Dev 2007. 16:355-60). However, it has been reported that inhibition of CD26 on CD34+ G-CSF mobilized peripheral blood (PB) cells does not improve migration in response to SDF-1 or engraftment into NOD/SCID mice (Kawai K, et al, et al. Stem Cells and Dev 2007. 16:361-70). Given the widespread usage of un-fractionated mobilized PB as a source of cells for transplantation in both the autologous and allogeneic setting, the implications of also studying un-fractionated mobilized PB should not to be overlooked. We therefore investigated whether inhibition of CD26 activity on mobilized PB (mobPB) mononuclear cells (MNC) would have an effect on CXCL12/SDF-1 mediated adhesion and migration. These results were compared directly with data from CB MNC. Non-adherent MNCs from mobPB and CB were obtained by density centrifugation followed by adhesion to plastic for 90 minutes. By flow cytometry, CD26 expression was approximately 48% and 20% of the CD45+ cell population with Mean Fluorescent Intensity (MFI) of 1,200 and 1,000 for mobPB and CB MNCs respectively. Expression of CD26 was confirmed by Western Blot in total cell lysates from mobPB and CB MNCs. CD26 activity, as determined using the substrate Gly-Pro P-nitroanilide (Gly-Pro-pNA), was 36.7±2.8 pmol pNA/minute for mobPB MNCs and 30.9±2.8 pmol pNA/minute for CB MNCs (n=16). Prior to use in functional in vitro assays, cells were first cultured with or without a 5mM concentration of the CD26 inhibitor Diprotin A (DpA) for 15 minutes in DPBS+0.2%BSA at 37oC, 5% CO2. To perform static adhesion assay, cells were treated with SDF-1α (0-800 ng/mL) for 30 minutes, loaded onto human fibronectin coated plates, and then allowed to incubate an additional 30 minutes at 37oC, 5% CO2. DpA pre-treatment of mobPB and CB MNC was observed to result in an increase in adhesion as compared to untreated cells. Specifically, the percent adherence to fibronectin in response to 400ng/mL SDF-1α increased from 50.1±4.5% for untreated to 70.1±2.3% for DpA treated mobPB MNCs (p≤0.05, n=6). The percent adherence for CB MNCs increased from 48.2%±5.1 for untreated to 65.4±5.9% for DpA treated cells in response to 400ng/mL SDF-1α (p≤0.05, n=6). To assess migration, chemotaxis assays were performed by loading 2×105 cells into the upper chamber of a transwell plate, adding SDF-1α (0-400 ng/mL) in the lower chamber, and incubating for 2 hours at 37oC, 5% CO2. DpA pre-treatment of mobPB and CB cells was observed to result in an increased migration as compared to untreated cells. Specifically, the percent migration at 400ng/mL SDF-1α was 20.0±1.7% and 27.8±1.9% for untreated and DpA treated mobPB MNCs respectively (p≤0.01, n=7). The percent migration in response to 400ng/mL SDF-1α was 58.8±1.4% for untreated CB MNCs and 70.6 ± 2.9% for DpA treated CB MNCs (p≤0.01, n=4). These data indicate that a substantial portion of G-CSF mobPB cells express CD26 and exhibit CD26 peptidase activity similar to that seen on CB cells. In addition, CD26 inhibition on these cells results in an increase in functional response to SDF-1 as quantitated by adhesion and migration. Taken in the context of other studies examining sorted CD34+ mobPB our data suggest that un-fractionated mobPB may have inherently different regulatory properties due to the presence of accessory cells. Additionally, our data suggest that suppression of functional response of HSC/HPC to the chemokines SDF-1 by CD26 activity may be an endogenous regulatory mechanism that extends beyond CB to also include mobPB. Future studies are needed to pursue whether inhibition of CD26 activity can serve as a technique by which the trafficking of hematopoietic stem and progenitor cells from un-fractionated mobilized peripheral blood can be manipulated for possible clinical benefit. Disclosures:No relevant conflicts of interest to declare.

CD26 Inhibitor Treated and CD26−/− Recipient Mice Exhibit Improved Transplant Efficiency as Quantitated by Increased Short-Term Homing and Long-Term Engraftment

A firm understanding of the biology of hematopoietic stem and progenitor cell (HSC/ HPC) trafficking is believed to be critical for the development of methodologies to improve transplant efficiency and subsequently immune reconstitution during hematopoietic stem cell transplantation in the clinical setting. Through the use of CD26 inhibitors and CD26 deficient mice (CD26−/−), we have previously generated data in mice suggesting that suppression of CD26/DPPIV (dipeptidylpeptidase IV) enzymatic activity on the transplant donor cell population can be utilized as a method of increasing transplant efficiency (Christopherson, KW 2nd, et al, Science 2004. 305:1000–3). However, the clinical importance of the transplant recipient should not to be overlooked given the potential importance of the bone marrow microenvironment in regulating the transplant process. We therefore investigated here whether inhibition or loss of CD26 activity in recipient mice would have an effect on transplant efficiency utilizing an in vivo congenic mouse model of transplantation. The short-term homing and long-term engraftment of BoyJ donor cells (expressing CD45.1+) into lethally irradiated control C57BL/6, CD26 inhibitor (Diprotin A) treated C57BL/6, or CD26−/− mice (expressing CD45.2+) was monitored by flow cytometric analysis of the bone marrow and peripheral blood at 24 hours and 6 months post-transplant respectively. Twenty-four hours post-transplant of 20×106 BoyJ mononuclear cells, we observed 8.85±0.58%, 10.69±1.01%, and 12.45±1.33% donor derived Sca-1+lin− cells in the bone marrow of recipient mice for control, Diprotin A treated, and CD26−/− recipient mice respectively. As compared to control mice, this represents a 20.8% increase (p=0.01) with CD26 inhibitor treatment and a 40.7% increase (p£0.05) resulting from the use of a CD26−/− recipient in short-term homing (N=5 mice per group). Six months post-transplant of 1×105 BoyJ mononuclear cells, we observed 39.90± 4.38%, 70.22± 3.72%, and 92.51± 1.04% donor contribution to hematopoiesis in the peripheral blood of control, Diprotin A treated, and CD26−/− recipient mice respectively. This represents a 76.0% increase (p£0.01) with CD26 inhibitor treatment and a 131.9% increase (p£0.01) as a result of the CD26−/− recipient in long-term engraftment as compared to control recipient mice (N=14 mice per group). These results provide pre-clinical evidence of the importance of CD26 expression within the transplant recipient with regard to regulating hematopoietic stem cell homing and engraftment. Our results also support the potential use of CD26 inhibitors to treat transplant patients during hematopoietic stem cell transplantation as a method of improving transplant efficiency. Lastly, our use of inhibitor treated C57BL/6 and CD26−/− recipient mice, which are also on a C57BL/6 background, in conjunction with a congenic model of transplantation provides a accurate and convenient model system for the in vivo testing of the efficacy of existing and new CD26 inhibitors in transplant recipients.

The Effects of Endomorphins on Striatal [3H]Gaba Release Induced by Electrical Stimulation: An In vitro Superfusion Study in Rats

The endomorphins (EM1 and EM2) are selective endogenous ligands for mu-opioid receptors (MOR1 and MOR2) with neurotransmitter and neuromodulator roles in mammals. In the present study we investigated the potential actions of EMs on striatal GABA release and the implication of different MORs in these processes. Rat striatal slices were preincubated with tritium-labelled GABA ([(3)H]GABA), pretreated with selective MOR1 and MOR2 antagonist beta-funaltrexamine and selective MOR1 antagonist naloxonazine and then superfused with the selective MOR agonists, EM1 and EM2. EM1 significantly decreased the striatal [(3)H]GABA release induced by electrical stimulation. Beta-funaltrexamine antagonized the inhibitory action of EM1, but naloxonazine did not affect it considerably. EM2 was ineffective, even in case of specific enzyme inhibitor diprotin A pretreatment. The results demonstrate that EM1 decreases GABA release in the basal ganglia through MOR2, while EM2 does not influence it.

72: Recipient Based Strategies for Improved Transplant Efficiency: CD26 Inhibitor Treated and CD26 -/- Recipient Mice Exhibit Increased Short-Term Homing and Long-Term Engraftment of Hematopoietic Stem and Progenitor Cells

Introduction: Through the use of CD26 inhibitors and CD26 deficient mice (CD26-/-), we have previously generated data suggesting that suppression of CD26/DPPIV activity on the transplant donor cell population could potentially be utilized clinically as a method of increasing transplant efficiency. However, the clinical importance of the transplant recipient should not to be overlooked. We therefore investigated whether inhibition or loss of CD26 activity in the recipient would have an effect on hematopoietic stem cell transplantation utilizing an in vivo congenic mouse model of transplantation. Methods: The short-term homing and long-term engraftment of BoyJ donor cells (expressing CD45.1+) into lethally irradiated control C57BL/6, CD26 inhibitor (Diprotin A) treated C57BL/6, or CD26-/- mice (expressing CD45.2+) was monitored by flow cytometric analysis of the bone marrow and peripheral blood at 24 hours and 6 months post-transplant. Results: Twenty-four hours post-transplant of 20 × 106 BoyJ mononuclear cells, we observed 8.85 ± 0.58%, 10.69 ± 1.01%, and 12.45 ± 1.33% donor derived Sca-1+lin- cells in the bone marrow of recipient mice for control, Diprotin A treated, and CD26-/- recipient mice respectively. As compared to control mice, this represents a 20.8% increase (p = 0.01) with CD26 inhibitor treatment and a 40.7% increase (p ≤ 0.05) resulting from the use of a CD26-/- recipient in short-term homing (N = 5 mice per group). Six months post-transplant of 1 × 105 BoyJ mononuclear cells, we observed 39.90 ± 4.38%, 70.22 ± 3.72%, and 92.51 ± 1.04% donor contribution to hematopoiesis in the peripheral blood of control, Diprotin A treated, and CD26-/- recipient mice respectively. This represents a 76.0% increase (p ≤ 0.01) with CD26 inhibitor treatment and a 131.9% increase (p ≤ 0.01) as a result of the CD26-/- recipient in long-term engraftment as compared to control recipient mice (N = 14 mice per group). Conclusions: These results provide pre-clinical evidence of the importance of CD26 expression within the transplant recipient with regard to regulating hematopoietic stem cell homing and engraftment. Our results also support the potential use of CD26 inhibitors to treat transplant patients during hematopoietic stem cell transplantation as a method of improving transplant efficiency.