Biol. Chem., Vol. 392, pp. 189–198, March 2011 • Copyright � by Walter de Gruyter • Berlin • New York. DOI 10.1515/BC.2011.002

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Expression and spatial heterogeneity of dipeptidyl peptidases

in endothelial cells of conduct vessels and capillaries

Veerle Matheeussen1, Lesley Baerts1, Guido DeMeyer2, Gilles De Keulenaer3, Pieter Van der Veken4,Koen Augustyns4, Veronique Dubois1, SimonScharpe1 and Ingrid De Meester1,*1 Laboratory of Medical Biochemistry, Department ofPharmaceutical Sciences, University of Antwerp,Universiteitsplein 1, B-2610 Wilrijk (Antwerp), Belgium2 Laboratory of Pharmacology, Department ofPharmaceutical Sciences, University of Antwerp,Universiteitsplein 1, B-2610 Wilrijk (Antwerp), Belgium3 Laboratory of Physiology, Department of PharmaceuticalSciences, University of Antwerp, Universiteitsplein 1,B-2610 Wilrijk (Antwerp), Belgium4 Laboratory of Medicinal Chemistry, Department ofPharmaceutical Sciences, University of Antwerp,Universiteitsplein 1, B-2610 Wilrijk (Antwerp), Belgium

* Corresponding authore-mail: ingrid.demeester@ua.ac.be

Abstract

Dipeptidyl peptidase IV (DPPIV)/CD26 is by far the mostextensively studied member of the prolyl oligopeptidase fam-ily of serine proteases. The discovery of the related enzymesDPP8 and DPP9 necessitates a (re-)evaluation of the DPPIV-like enzymatic activity in cells and organs. In this study, weaimed (1) to investigate the expression of the individualdipeptidyl peptidases in different types of endothelial cells(ECs) and (2) to reconsider published data in relation to ourfindings. Examination of DPP expression in rat primary ECsof aortic, endocardial and cardiac microvascular originrevealed the presence of DPPIV-like activity in all celllysates. More than half of this activity could be attributed toDPP8/9. Western blot analysis revealed an abundance of theDPP8 protein as compared to DPP9. The expression ofDPPIV and DPP8 was significantly higher in the cardiacmicrovascular endothelium than in the other ECs, suggestinga more pronounced role of these DPPs in the microvascu-lature. In situ, DPP activity in ventricular microvasculaturewas completely inhibited by sitagliptin, indicating thatDPPIV is the predominant DPPIV-like enzyme in this organ.By contrast, immunohistochemical studies indicated DPP9 asthe predominant DPP in human carotid artery ECs. In con-clusion, our results support a highly regulated expression ofindividual DPPs in ECs, with a spatial heterogeneity in thecardiovascular tree.

Keywords: CD26; DPP4; endothelial heterogeneity;endothelium; vasculature.

Introduction

Dipeptidyl peptidase IV/CD26 (DPPIV) is a cell-surfaceprotease that belongs to the prolyl oligopeptidase family ofserine proteases. It cleaves off N-terminal dipeptides frompeptides with proline or alanine at the penultimate position.Discovered in 1966 (Hopsu-Havu and Glenner, 1966), it isby far the most extensively studied member of this familyof serine proteases and is clinically important as therapeutictarget for the treatment of type II diabetes (Lambeir et al.,2003). The discovery of the related enzymes DPP8 andDPP9 (Abbott et al., 2000) necessitated a (re-)evaluation ofthe expression of the dipeptidyl peptidase family members.For a long time the DPPIV-like enzymatic activity in cellsand organs was attributed solely to DPPIV, whereas DPP8and DPP9 have very similar kinetic profiles towards the dif-ferent substrates used for DPPIV activity measurements.Moreover, their activity can also be inhibited by non-selec-tive DPP inhibitors (Bjelke et al., 2006).

Recently, several research groups have investigated thedistribution of the cytosolic enzymes, DPP8 and DPP9, intissues and cells at protein and/or activity level. DPP8/9activity has been reported in human brain (Stremenova et al.,2007; Busek et al., 2008), in bovine and rat testes and epi-didymis (Dubois et al., 2009), rat lung (Schade et al., 2008)and rat brain (Frerker et al., 2007). In mice, DPP8/9 activitywas detected in the immune system, brain, testis, muscle(skeletal, heart and uterine) and epithelium of colon, lungand liver (Yu et al., 2009). DPP8/9 activity was also foundin human peripheral blood mononuclear cells and in the celllines Jurkat, U937, Raji, 293T, Colo-16, 3T3, Huh-7, HepG2and CHO (Maes et al., 2007; Yu et al., 2009).

Our interest in DPPs in endothelial cells (ECs) wasstrengthened following several recent findings. First, DPPIVinhibition (as add-on to G-CSF treatment) improved cardiacfunction in an acute myocardial infarction model in mice.Prolongation of the biological half-life of SDF-1a

(CXCL12), a natural substrate of DPPIV, is proposed as theresponsible mechanism (Zaruba et al., 2009). A clinical trialis ongoing to test the safety and efficacy of this strategy inpatients (Theiss et al., 2010). Second, a beneficial effect ofDPPIV inhibition in the treatment of heart failure has beenpostulated, owing to the increased biological half-life ofanother substrate of DPPIV, namely BNP(1–32) (Brandt etal., 2006; Vanderheyden et al., 2009). Finally, we reported adecrease in ischemia/reperfusion injury, in rat and mouselung transplantation models, by flushing and storage of thegraft in a solution with the irreversible DPP inhibitor, AB192(Belyaev et al., 1999; Zhai et al., 2007; Jungraithmayr et al.,2010). Each of these observations suggests an involvementof vascular DPPs.

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Given the evidence for extensive endothelial heterogene-ity, it is not surprising that the expression of a defined proteincan differ among different animals and organs (Bicknell,1996). Even in the same organ, the endothelium of large andsmall vessels, veins and arteries exhibits significant hetero-geneity (Garlanda and Dejana, 1997) and the existence of adifferent intrinsic genetic program for endocardial, aortic andcardiac microvascular ECs has been hypothesized (Hendric-kx et al., 2004).

DPPIV is present in the capillary bed of most organs inhumans and laboratory animals (Table 1). Concerning thepresence of DPPIV in conduct vessels, contradictory resultsare found in the literature. Most often, only the capillaryendothelium showed DPPIV reactivity, whereas conduct ves-sels were negative (Gossrau, 1979; Lojda, 1979). However,at least two research papers reported the presence of DPPIVin arteries of spleen and lung (Hartel et al., 1988; Hartel-Schenk et al., 1990). In cell culture experiments, DPPIVpresence was described in microvascular as well as macro-vascular ECs (Table 2).

Very little is known about the presence of DPP8 and DPP9in endothelial cells. In baboon tissues, DPP8 mRNA wasfound in the endothelium in small vessels of an inflamedpneumonic lung, in the endothelium of the skeletal muscleand in the capillary endothelial cells of the colon. However,DPP8 or DPP9 mRNA was not present in sinusoidal endo-thelium of the spleen (Yu et al., 2009). The protein expres-sion or activity levels of DPP8 and DPP9 in endothelial cellshave not been reported so far. Therefore, we investigated inthe present study the expression of different DPPs in primaryisolated endothelial cells from rat heart (endocardial and car-diac microvascular EC) and aorta (aortic EC), and in situ, inthe endothelium of rat heart and aorta and human carotidartery specimens.

Results

DPP protein expression in primary rat endothelial

cell lysates

DPPIV, DPP8 and DPP9 protein expression was studied inthree different types of rat ECs by Western blot analysis. Theidentity of the isolated and cultured ECs was confirmed mor-phologically by their cobblestone-like appearance and by theprotein expression of the endothelial marker, CD31 (alsoknown as PECAM-1 or platelet/endothelial cell adhesionmolecule-1) (Figure 1A).

The three enzymes of interest could be detected in thedifferent types of ECs (Figure 1A). Comparison of theirexpression levels revealed a higher amount of DPPIV andDPP8 protein in the cardiac microvascular endothelial cells(CMVE) as compared to the aortic and endocardial endothe-lial cells (AE and EE). A very low and comparable expres-sion of the DPP9 protein was detected in all cell types.

The Western blot analysis was repeated three times, usingcell lysates from three different cell isolations, with similarresults. Expression levels were quantified, all normalized tothe b-actin levels, and all compared to the protein levels in

CMVE (Figure 1B). Expression of DPPIV and DPP8 wasapproximately 60% lower in AE and EE compared to CMVE(p-0.005). DPP9 protein levels were equal for the differentEC types.

Western blot detection of DPPIV in the EC lysatesrevealed a band migrating at the same molecular mass as thepurified rat kidney DPPIV (Schmauser et al., 1999), DPP8migrated at lower and DPP9 at higher molecular mass aspreviously detected with the same antibodies for recombinanthuman DPP8 and purified bovine DPP9 (Dubois et al.,2009), but at the same molecular mass as the band detectedin rat testes homogenate (data not shown).

Specific DPP activity in primary rat endothelial

cell lysates

In addition to the DPP protein expression, specific dipeptidylpeptidase activity was examined in cell lysates of the differ-ent types of rat ECs. DPP activity could be measured in allthe different endothelial cells that were studied (Figure 2),which is in overall agreement with the Western blot results.As measurements were performed at pH 8.3, total DPP activ-ity represents the combined activities of DPPIV, DPP8 andDPP9. At this pH DPPII is not active (Maes et al., 2005). Incontrast to the Western blot analysis, activity measurementscannot make a distinction between DPP8 and DPP9 becauseno selective inhibitors have been developed yet. DPPIVactivity is defined as the part of the total DPP activity thatcan be inhibited by sitagliptin and DPP8/9 as the part inhib-ited by allo-Ile-isoindoline.

In all three different EC lysates, more than half of the totalDPP activity could be attributed to DPP8/9 enzymatic activ-ity. In accordance with the highest expression of DPPIV andDPP8 protein, a significantly higher total DPP activity andalso DPPIV and DPP8/9 activity is found in CMVE com-pared to AE and EE. It is remarkable that the two latter typesof ECs contain very similar enzymatic DPP activities (Figure2), as well as identical protein expression levels of DPPIV,DPP8 and DPP9 (Figure 1).

Specific DPP activity in rat heart and aorta

homogenates

To determine whether the enzyme activity in the ECs isresponsible for most of the DPP activity in the organs fromwhich the cells were isolated, DPP activity was measured inhomogenates from rat heart and rat aorta (Figure 2). Specifictotal DPP activities in the homogenates were lower as com-pared to the endothelial cell lysates and the attribution of thedifferent enzymes to the total DPP activity was shifted frommainly DPP8/9 activity in ECs (;60% of total activity) topredominantly DPPIV enzymatic activity in the organs(;80% of total activity in heart and ;90% in aorta).

DPP activity in rat heart and aorta, studied by a

histochemical enzyme activity assay

DPP activity could be detected in the capillaries of the ratheart, whereas no activity was found in the endocardial endo-

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Table 1 DPPIV expression in endothelium: a literature overview of histochemistry experiments.

Species Methoda Endothelial DPPIV positive Endothelial References

ACT IHCorgans/systems cell type

Mice Brain: positivity in certain brain regions Capillary Schnabel et al., 1988Mice 6 Brain: blood-brain barrier regions and Capillary Schnabel et al., 1992

weak staining in somecircumventricular organs

Rat 6 Kidney: low intensity in capillary Capillary Fukasawa et al., 1981endothelium of the glomeruli, nostaining of capillary endothelium indistal and collecting tubules

Rat 6 Thyroid gland: clear staining, faint Capillary Sahara et al., 1981staining: parathyroid gland, pinealgland, anterior and posterior pituitarygland and pancreasAdrenal gland: cortex Sinusoid

Rat 6 Lung: capillary bed of the alveolar septa Capillary Krepela et al., 1985Rat 6 Skeletal muscle: venous part of the Capillary Mrazkova et al., 1986

capillary bedRat 6 Submandibular, sublingual and Capillary Hartel et al., 1988

extraorbital gland, tongue, skin, larynx,heart, lung, stomach, pancreas,medullary region of the kidney, uterus,fallopian tube, ovary, cerebrum andcerebellumLiver, adrenal cortex, spleen, ovary, Sinusoidcorpus luteumMany, e.g., spleen, but not all organs Artery

Rat 6 Liver Not specified Fukui et al., 1990Rat 6 6 Heart, liver, lung, spleen, esophagus, Capillary Hartel-Schenk et al., 1990

tongue, fat tissue, cerebrum andcerebellumLiver SinusoidLung Vein artery

Rat 6 Kidney: medulla, spleen: red pulp, Not specified McCaughan et al., 1990cerebellum, stomach, heart and skeletalmuscle

Rat 6 Brain: only in choroid plexus Capillary De Bault and Mitro, 1994Rat 6 Heart Capillary Koyama et al., 1998Rat 6 Heart: left ventricular tissue Capillary Okruhlicova et al., 2005Rabbit 6 Cornea: corneal stroma Capillary Cejkova et al., 2004Pig 6 Pancreas: on the luminal side of Capillary Poulsen et al., 1993

capillariesRodents, cat, 6 Different organs Capillary Gossrau, 1979humanRat 6 Most organs: venous part of the Capillary Lojda, 1979Mini-pig capillary bed, not in brainRabbit DPP activity was confined to vasaCock vasorum, no reaction in the aorticHuman endothelium

Similar to arteries: only positivity in apart of the vasa vasorum

Laboratory 6 Skeletal muscle: venous part of the Capillary Grim and Carlson, 1990animals, human capillary bed

Skeletal muscle VeinRodents 6 Nasal cavity: venous part of the Capillary Kubisova and Pospisilova, 1992Human capillary bedPig 6b 6 Ileum: lamina propria as well as all Capillary Hansen et al., 1999Human other layers of the intestinal wall

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Table 1 (Continued)

Species Methoda Endothelial DPPIV positive Endothelial References

ACT IHCorgans/systems cell type

Human 6 6 Placenta, liver, salivary glands, Vascular Feller et al., 1986pancreas and kidney Lymphatic

Human 6 Dental pulp Not specified Dubovy and Kukletova, 1992Human 6 Nasal mucosa: weak staining Capillary Grouzmann et al., 2002Human 6 6 Brain Capillary Stremenova et al., 2007Human 6 Bronchial mucosa: very low reactivity Not specified Landis et al., 2008aACT: activity staining with glycyl-prolyl-methoxy-naphtylamide and Fast Blue B on cryostat sections; IHC: immunohistochemistry.bActivity staining with alanyl-prolyl-methoxy-naphthylamide.

Table 2 DPPIV expression in endothelium: a literature overview of cell culture experiments.

Species Cells Analysis References

Activitya Proteinb Genec

Mouse Lung microvascular ECs 6 6 Favre et al., 2003Rat Lung microvascular ECs 6 Johnson et al., 1993Rat Lung microvascular ECs 6 Cheng et al., 1998Rat ECs isolated from rat heart 6 Koyama et al., 1998Pig Aortic ECs 6 Palmieri and Ward, 1989Human HUVECs 6 Ghersi et al., 2001Human HUVECs, neonatal foreskin microvascular ECs 6 Aimes et al., 2003Human Human glomerular ECs 6 6 6 Pala et al., 2003Human HUVECs 6 6 Silva et al., 2003Human HUVECs, dermal microvascular EC line 6 6 Eltzschig et al., 2006Human HUVECs 6 6 Ghersi et al., 2006Human Primary dermal lymphatic ECs, blood vascular ECs 6 6 6 Shin et al., 2008Human Bone marrow EC line 6 Sun et al., 2008Human aortic ECs, microvascular dermal ECs 6 6 6 Pala et al., 2010aActivity measurements on cell lysates or whole cells.bWestern blotting, ELISA or immunocytochemistry.c(Real-time) RT-PCR or oligonucleotide microarrays.ECssendothelial cells; HUVECsshuman umbilical vein endothelial cells.

Figure 1 DPP protein expression levels in primary isolated endothelial cells.(A) Western blot analysis of cell lysates from three different types of endothelial cells: cardiac microvascular endothelium (CMVE),endocardial endothelium (EE) and aortic endothelium (AE). Detection of the endothelial cell marker CD31 and the proteins of interestDPPIV, DPP8 and DPP9. (B) Quantification of the Western blot results from panel (A). All values are normalized to the b-actin signal andpresented as the percentage of expression of the DPP protein compared to the expression level in cardiac microvascular endothelial cells(CMVE). The differences in DPP protein level between the cell types were assessed with a one-sample test, with a test value of 100.*p-0.005 versus CMVE (100%).

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Figure 2 DPP-specific enzymatic activity in primary isolatedendothelial cells: cardiac microvascular endothelium (CMVE),endocardial endothelium (EE) and aortic endothelium (AE) and inrat heart and aorta homogenate.DPPIV and DPP8/9 activities in the cell lysates were calculatedbased on the inhibition profiles using sitagliptin for DPPIV and allo-Ile-isoindoline for DPP8/9. Specific activities (U/g protein) are rep-resented as the mean"standard deviation of three separateexperiments. Differences in DPP activity levels between the endo-thelial cells were analyzed with factorial analysis variance withduplicates (two levels) as within subject factor and cell type (threelevels) as between subject factor. The Bonferroni test was used aspost hoc test. **p-0.005, *p-0.01, versus CMVE.

Figure 3 In situ expression of DPPs in rat heart and human carotid arteries.(A) Detection of DPP activity with a histochemical activity assay in rat heart. Cryostat sections were incubated with glycyl-prolyl-hydroxy-naphtylamide and nitro blue tetrazolium without (left) or in the presence (right) of sitagliptin. The arrow indicates the endocardial endothe-lium. Bar sizes100 mm. (B) Cryostat sections of human carotid arteries from vascular surgery (endarterectomy) or autopsy, stained for theendothelial cell marker CD31 and the proteins of interest DPPIV, DPP8 and DPP9. Predominantly DPP9 is present in the endothelium ofthese conduct vessels. Bar sizes100 mm.

thelium and in the aortic endothelium (Figure 3A). Becauseall the activity could be inhibited by sitagliptin, the DPPactivity could be attributed to DPPIV. DPP8/9 activity couldnot be measured.

DPP protein expression in endothelium of human

carotid arteries, studied by immunohistochemistry

The immunohistochemical study of DPPIV, DPP8 and DPP9expression in the endothelial cells of human carotid arteriesfrom endarterectomy and autopsy specimens revealed for thefirst time DPP9 as the only detectable DPPIV-like enzymein the endothelium of these conduct vessels (Figure 3B).However, not all ECs were DPP9 positive.

Discussion

The discovery of the DPPIV-related enzymes, DPP8 andDPP9, imposes a re-evaluation of the expression profiles ofthe dipeptidyl peptidase family members in organs and cells.Here, we focused on the DPP expression in cardiac andmacrovascular endothelium. DPPIV, DPP8 and DPP9 weredetected on protein and activity level in primary isolated rataortic (AE), endocardial (EE) and cardiac microvascularendothelial cells (CMVE). A more pronounced expression ofboth DPPIV and DPP8 protein was present in CMVE ascompared to AE and EE. DPP9 protein expression was equalin the different EC types. The combined data from the West-ern blot analysis and the enzyme activity measurements sug-gest that mainly DPP8 is responsible for the DPP8/9 activitybecause of the high amount of DPP8 protein as compared toa low DPP9 expression. However, taking into account thatactivity of DPP8 seems highly unstable in nature (ProtParam,

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ExPASy tool; Gasteiger et al., 2005), it cannot be excludedthat at least part of the enzyme is already occurring in acatalytically inactive form in the cytosol or that a part of theactivity of the enzyme is lost during manipulations prior tothe activity assays. In addition, the DPP8 specific antibodyused is directed against the N-terminal end of the protein,whereas the catalytic region is located in the C-terminalregion (Bjelke et al., 2006). However, even if both regionsare present, enzymatic activity is not guaranteed. Our obser-vations underscore the need for the development of selectiveDPP8 and DPP9 inhibitors, as a tool to investigate the enzy-matic activity of each of these dipeptidyl peptidases sepa-rately in their physiological context.

In the present study, DPP8 and DPP9 protein were detect-ed at a molecular mass of 84 kDa and 120 kDa, respectively.The recombinant proteins are known to migrate at100–111 kDa (DPP8) and 98 kDa (DPP9) (Olsen and Wagt-mann, 2002; Chen et al., 2004). Detection of a shorter formof DPP8 has been reported earlier, in cell lysates of humanHeLa cells. Whether this band represents a splice variant ora proteolysis product of DPP8 is still unknown (Geiss-Fried-lander et al., 2009). DPP9 migration at a higher molecularmass has not been described before, probably because dataon rat DPP9 protein are still very scarce. There is some evi-dence for a longer form of human DPP9 of 971 amino acids(association number NCBI AF542510) compared to the ‘full-length’ DPP9 of 892 amino acids (Ajami et al., 2004).

With regard to DPPIV our results are consistent with pre-vious observations. For example, total DPP activity presentin rat AE (1.03 U/g) (Figure 2) corresponds to a previouslyreported experiment where a DPP activity of 1.5 U/g wasmeasured in pig primary isolated AE (Palmieri and Ward,1989). The higher DPPIV protein expression in microvas-cular ECs described here corroborates the higher gene levelof this enzyme in human neonatal foreskin microvascularECs compared to human umbilical vein endothelial cells(HUVECs) (Aimes et al., 2003). Other research groups alsofound that the expression of DPPIV is regulated in a cell-type specific manner (microvascular versus macrovascularendothelium) (Aimes et al., 2003; Pala et al., 2010). Theseobservations all suggest a role of DPPIV in regulation ofcapillary exchange and in local modulation of substratesrather than an influence on the vascular tone.

We preferred primary culturing of the ECs and harvestingafter the first passage above the use of cell lines and multiplepassages for many reasons. Rapid loss of endothelial char-acteristics of primary isolated ECs has been described, prob-ably owing to the production of interleukin-1a (Maier et al.,1990). The many attempts to immortalize ECs from varioussources have been disappointing for the main reason thatupon immortalization endothelial cells frequently lose theirendothelial characteristics and show greater similarity tofibroblasts (Bicknell, 1996). Although cell culture experi-ments have limitations, they remain relevant in the search forthe physiological role of many proteases. Observation of cul-ture-induced changes can reflect the ability of diseased cellsto adapt by altering their protein expression (Sandow andGrayson, 2009). Cultured cells are a useful tool for studying

changes in protein expression upon exposure of differenttypes of ECs to distinct (patho-)physiological stimuli, where-as histochemical studies can add value when extrapolatingthe in vitro observations to the in vivo situation.

Histochemical DPP activity staining in rat heart and aortarevealed the presence of DPPIV activity in capillaries of therat heart and the absence in cardiac endothelium and rat aor-ta. This observation confirmed earlier reports of predominantDPPIV expression in the capillary bed of organs (Lojda,1979). DPP8/9 activity could not be detected in the capillarybed of the heart. To our knowledge, in situ DPP8/9 activityhas only been reported in lung parenchyma. Results wereobtained after a very long incubation of 20 h (Schade et al.,2008).

In our study, the absence of DPP activity in situ in endo-cardial and aortic endothelium did not match the observa-tions in cultured cells, where DPP activity could be measuredboth in EE and AE. Discrepancies of results in histochem-istry of fresh tissues versus cell culture experiments are com-mon (see Table 1 versus Table 2). Although most researchgroups report the presence of DPPIV predominantly in thecapillary bed of most organs and not in conduct vessels, cellculture experiments detect DPPs both in microvascular andmacrovascular ECs (e.g., HUVECs and aortic ECs). Upre-gulation of DPPs during EC isolation and culturing is themost probable cause of this discrepancy.

By using immunohistochemical stains, we showed for thefirst time the expression of DPP9 protein in discrete zonesof human carotid artery ECs. The absence of DPPIV in theseconduct arteries corroborates earlier findings (Lojda, 1979)and was expected as we observed a lower DPPIV proteinexpression and activity level in ECs isolated from conductvessels.

To gain insight into the contribution of the DPPs in theendothelium to the total DPP activity in a particular organ,DPP activities in rat heart and aorta homogenates were meas-ured (Figure 2). Specific total DPP activity was lower in thehomogenates compared to the EC lysates and the attributionof the different enzymes to the total activity was reversed.The activity in the rat heart homogenate is comparable tomeasurements in mice hearts, where the total DPP activitydecreased with approximately 20% by adding a DPP8/9inhibitor (Yu et al., 2009). In rat heart homogenates, DPP8/9also represented approximately 20% of the total DPP activity.

Previously, it was postulated that the contribution ofDPPIV activity of the capillary endothelium to the totalDPPIV activity was decisive in the myocardium and aorta(Lojda, 1979). The method used to prove this statement wasan activity staining on cryostat sections with glycyl-prolyl-methoxy-naphtylamide as a substrate and 10-3 M diisopropylfluorophosphates (DFP) as inhibitor. It is obvious that thenon-selective irreversible serine-protease inhibitor, DFP, alsoinhibits enzymatic activity of DPP8 and DPP9 on tissue slic-es. The activity staining that we performed in this study canattribute the activity exclusively to DPPIV by using theselective DPPIV inhibitor, sitagliptin. When comparingobtained results with previously reported histochemistryexperiments (Table 1) it should be kept in mind that a lot of

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the activity staining was performed before the discovery ofDPP8 and DPP9 and even in the years thereafter specificDPPIV inhibitors were not used to assign the observed activ-ity to DPPIV.

In conclusion, we report here for the first time the proteinexpression and activity levels of DPP8 and DPP9 in rat pri-mary isolated ECs. We also confirm earlier reports about thepredominant expression of DPPIV in capillaries, comparedto conduct vessels. In the myocardium, DPPIV was the mainenzyme responsible for DPP activity and was located in cap-illaries. Finally, DPP9 was found to be the predominant DPPin endothelium of conduct vessels.

Materials and methods

Tissue homogenates

Heart, aorta and testis were removed from the Wistar rat(150–200 g), washed in PBS and snap frozen in liquid nitrogen.Heart and aorta were homogenized in enzyme activity lysis buffer(1% octylglucoside, 10 mM EDTA and 70 mg/ml aprotinin in a50 mM Tris buffer, pH 8.3), the testis in Western blot lysis bufferw1% Triton X-100, 150 mM NaCl and a complete protease inhibitorcocktail tablet (Roche Diagnostics, Vivoorde, Belgium) in a 50 mM

Tris buffer, pH 7.6x. After homogenization the tissue suspensionswere stirred end-over-end during 30 min at 48C, three times centri-fuged at 12 000 g for 30 min and finally 60 min at 18 000 g at48C. The final supernatant was used for enzyme activity measure-ments or Western blot analysis.

Rat primary endothelial cell isolation (AE and EE)

Aortic endothelial cells (AE) and endocardial endothelial cells (EE)were isolated using the isolation method as described previously(Hendrickx et al., 2004) with modifications. Briefly, adult male Wis-tar rats (150–200 g) were sacrificed by carbon dioxide gas and theheart and thoracic aorta were rapidly excised and placed in PBS.To obtain AE, the aorta was rinsed with PBS and dissected to exposethe luminal surface. After which the aorta was incubated with a0.2% collagenase solution at 378C during 1 h and the ECs werescraped free from the underlying tissue, centrifuged and resuspen-ded in DMEM culture medium (Lonza, Verviers, Belgium) supple-mented with 10% FBS, 2 mM glutamine, 100 U/ml penicillin and100 mg/ml streptomycin (Invitrogen, Merelbeke, Belgium). After4 h of incubation, the medium with contaminating cells was dis-carded and the attached ECs were incubated with fresh medium.Cell cultures were kept in a humidified incubator with 5% CO2 at378C. Culture medium was replaced every 2–3 days. To isolate EE,atria were removed from the heart and ventricles were cut open,washed with PBS and incubated with a 0.2% collagenase solutionat 378C during 15 min. EE were gently scraped, collected by cen-trifugation and further processed as described above.

Rat primary endothelial cell isolation (CMVE)

Cardiac microvascular ECs were isolated using a slightly modifiedpublished method (Nishida et al., 1993). Briefly, adult male Wistarrats were euthanized with carbon dioxide gas, the heart wasremoved and placed in a Krebs-Henseleit bicarbonate buffer. Afterremoval of connective tissue, the atria, the right ventricle and allvalvular tissue, the remaining left ventricular tissue was opened and

washed with buffer. Epicardial mesothelial cells and endocardialendothelial cells were devitalized by the exposure to 70% ethanolduring 30 s. The outer one-fourth of the ventricular wall was dis-sected away and the remaining tissue was minced finely and incu-bated with a 0.2% collagenase solution at 378C during 30 min ofshaking. Trypsin (0.02%) was added and the ventricular tissue frag-ments were sheared 10 times in a 10-ml pipette. After another incu-bation period of 30 min at 378C the dissociated cells were filteredthrough a 100-mm mesh. Cells were centrifuged and resuspendedin DMEM with 20% FBS and further treated as described in thesection above. Cells were lysed after the first passage.

Cell lysis

Washed cells were suspended in lysis buffer for enzyme activityassay (1% octylglucoside, 10 mM EDTA, and 70 mg/ml aprotininin a 50 mM Tris buffer, pH 8.3) or lysis buffer for Western blotanalysis w1% Triton X-100, 150 mM NaCl and a complete proteaseinhibitor cocktail tablet (Roche Diagnostics) in a 50 mM Tris buffer,pH 7.6x, incubated for 1 h and centrifuged for 10 min at 12 000 gat 48C. Protein content of the cell lysates was determined accordingto Bradford (1976) with BSA as a standard.

Enzyme activity assay

DPP activity in the EC lysates and tissue homogenates was meas-ured by using 0.5 mM glycyl-prolyl-methoxy-naphtylamide (Gly-Pro-MNA) (Sigma-Aldrich, Bornem, Belgium) as a fluorogenicsubstrate in a 50 mM Tris buffer pH 8.3. DPP activity was deter-mined kinetically during 10 min at 378C by measuring the initialvelocities of MNA release (lexs340 nm, lems430 nm) from thesubstrate using an Infinite� 200 reader (Tecan Benelux, Mechelen,Belgium). One unit of enzymatic activity (U) is the amount ofenzyme that catalyzes the release of 1 mmol MNA from the sub-strate per minute under the assay conditions (Scharpe et al., 1988).Under these conditions, DPPIV, DPP8 and DPP9 can cleave thesubstrate. A distinction between the DPPIV and DPP8/9 activitywas made by adding selective inhibitors: 10 mM sitagliptin (selec-tive DPPIV inhibitor) (Kim et al., 2005) and 5 mM allo-Ile-isoin-doline (selective DPP8/9 inhibitor) (Lankas et al., 2005) aspreviously described for a colorimetric enzyme activity assay(Dubois et al., 2009).

Western blot analysis

Cell lysates were separated on SDS-PAGE (7.5%) and the proteinswere transferred to a nitrocellulose membrane by electroblotting.After blocking with 5% milk powder in a 0.05 M Tris buffer during1 h at room temperature, blots were incubated with the primaryantibody during 1 h at room temperature and overnight at 48C. Allprimary antibodies were obtained from Abcam (Cambridge, UK):rabbit polyclonal antibody ab28340 against the spacer region ofDPPIV, ab42080 against the catalytic domain of DPP9 and ab42075against the amino terminal end of DPP8 and mouse monoclonalantibody ab24590 against CD31. Goat anti-rabbit and goat anti-mouse antibodies conjugated with HRP (Biosource, Merelbeke, Bel-gium) were used as secondary antibody with an incubation time of2 h at room temperature. Antibody detection was accomplished withan enhanced chemiluminescent substrate (Supersignal West Femtochemiluminescent substrate, Pierce, Rockford, IL, USA) and signalswere visualized using the OptiGo (Isogen, Sint-Pieters-Leeuw, Bel-gium). Blots were stripped and b-actin (mouse monoclonal anti-

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body, Sigma-Aldrich) was detected for normalization of the signals.Quantification of the images was performed using the TotalLab soft-ware (Isogen, Sint-Pieters-Leeuw, Belgium). Positive controls forthe primary antibodies were performed previously (Dubois et al.,2009).

Histochemical enzyme activity assay

The assay was performed according to a previously described meth-od (Dubois et al., 2009), with a substrate (glycyl-prolyl-hydroxy-naphtylamide) incubation period of 1 h.

Immunohistochemistry

Human carotid artery specimens were obtained from autopsy or vas-cular surgery. Autopsy specimens were collected within 24 h afterdecease, whereas surgery specimens were processed immediately.The samples were snap-frozen in optimal cutting temperature com-pound, immediately after removal. Frozen sections were cut at5–6 mm thickness. Human kidney and bovine testes were used asa positive control for the primary antibodies. These organs werehandled the same way as the human arteries. The kidney was usedas a positive control for the DPPIV antibody, bovine testis was usedto check the suitability of the DPP8 and DPP9 antibody both gen-erated against the human enzymes. DPPIV was clearly detected inthe brush border epithelial cells of the proximal tubules of the kid-ney (Feller et al., 1986), DPP8 in ‘early’ spermatozoids and DPP9in ‘later stage’ spermatozoids embedded in the epithelium of thetestes (Dubois et al., 2009). Frozen sections were fixed in acetonefor 5 min. Thereafter specimens were treated with 3% H2O2 toquench endogenous peroxidase activity and blocked with horseserum. Subsequent sections were incubated overnight with theappropriate primary antibodies. Immunohistochemical detection wasperformed with secondary biotin-labeled anti-rabbit/mouse antibod-ies in combination with streptavidin HRP and aminoethylcarbazole(red staining). The following primary antibodies were used: TA5.9mouse monoclonal antibody against human DPPIV (De Meester etal., 1993), Ab42076 rabbit polyclonal antibody against the spacerregion of DPP8 (Abcam), the non-commercial rabbit polyclonalantibody against a DPP9 sequence (Dubois et al., 2009) and themonoclonal mouse anti-human CD31 antibody (Dako, Glostrup,Denmark).

Statistical analysis

All values are reported as mean"standard deviation. Differences inDPP activity levels were assessed with factorial analysis variancewith duplicates (two levels) as within subject factor and cell type(three levels) as between subject factor. The Bonferroni test wasused as post hoc test. Differences in protein expression levelsbetween the different types of ECs were tested with a one-sampletest with a test value of 100. All analyses were performed with SPSSstatistics 16.0 software (SPSS Inc., Chicago, USA).

Acknowledgments

This work was supported by the Fund for Scientific Research Flan-ders (Belgium, FWO-Vlaanderen, grant no. G016209) and by BOFGOA UA (Research Council, Special Fund for Research, Universityof Antwerp). V.M. is a research assistant and P.V.d.V. is a postdoc-toral fellow of FWO-Vlaanderen. We are grateful to Mrs. NicoleLamoen and Mrs. Rita Van Den Bossche for their excellent technicalassistance.

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Received July 2, 2010; accepted August 16, 2010

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