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Published in final edited form as: Life Sci. 2015 Nov 26;159:83–89. doi: 10.1016/j.lfs.2015.11.026

Linagliptin Attenuates Diabetes-Induced Cerebral Pathological Neovascularization in a Blood Glucose-Independent Manner: Potential Role of ET-1

Mohammed Abdelsaid 1,3, Raeonda Williams 3, Trevor Hardigan 3, Adviye Ergul 1,2,3
PMCID: PMC4882278  NIHMSID: NIHMS743112  PMID: 26631506

Abstract

Aims

We have shown that glycemic control with metformin or endothelin-1 (ET-1) inhibition with bosentan prevents and restores diabetes-mediated cerebral pathological remodeling and neovascularization. Our recent data suggest that linagliptin, a member of the dipeptidyl peptidase-4 inhibitor class of glucose-lowering agents, prevents cerebrovascular remodeling and dysfunction independent of its blood glucose lowering effects. We hypothesized that linagliptin prevents pathological neovascularization via the modulation of the ET-1 system.

Materials and Methods

24-week old diabetic Goto-Kakizaki and nondiabetic Wistar rats were treated for 4 weeks with either vehicle chow or chow containing 166mg/kg linagliptin. At termination, FITC-dextran was injected to visualize the vasculature. Brain sections were imaged by confocal microscopy for vascular density, tortuosity, vascular volume, and surface in both the cortex and striatum. Retinal acellular capillary formation was measured. Brain microvascular endothelial cells (BMVEC) isolated from control or diabetic rats were treated with linagliptin with or without ET-1 dual receptor antagonist and tested for angiogenic properties with cell migration and tube formation assays.

Key Finding

Linagliptin reduced all indices of cerebral neovascularization compared with control rats. In vitro, linagliptin normalized the augmented angiogenic properties of BMVECs isolated from diabetic animals and bosentan reversed this response. Cells from diabetic animals had higher ET-1 and less ETB receptors than in control cells. Linagliptin significantly decreased ET-1 levels and increased ETB receptors.

Significance

ET system contributes to pathological neovascularization in diabetes as evidenced by restoration of functional angiogenesis by bosentan treatment and prevention of linagliptin-mediated improvement of angiogenesis in the in vitro model.

Keywords: Diabetes, Endothelin, Linagliptin, Neovascularization

Introduction

Diabetes and associated complications are growing at an alarming rate (1). Vascular disease lies at the heart of all diabetic complications, which are traditionally classified as either microvascular (kidney and eye) or macrovascular (heart and brain) (2, 3). However, it is increasingly recognized that cerebral microvascular disease is a major player in cerebral complications of diabetes such as increased risk and poor recovery of stroke as well as cognitive impairment. Regulation of cerebrovascular function and structure is critical for the maintenance of cerebral blood flow which ultimately is the most important determinant of brain function including cognition. We have shown that diabetes mediates pathological neovascularization in the brain just like in diabetic retinopathy (4–6). While there is an increase in vascular density and volume suggesting there is increased angiogenesis and remodeling of the existing vessels, these vessels demonstrate less pericyte coverage and increased permeability. Since large scale clinical trials showed that tight glycemic control prevents/reduces microvascular complications of diabetes (7, 8), in our previous studies we investigated the impact of glycemic control with metformin on cerebral microangiopathy. We have shown that metformin was effective in preventing and reversing dysfunctional neovascularization (5, 9). Given that metformin has direct antioxidant effects, whether the observed changes were due to glycemic control or direct effects of metformin remained unresolved (10).

Endothelin-1 (ET-1), a potent vasoactive peptide produced by endothelial cells, has emerged as a critical mediator of tumor angiogenesis, another form of pathological angiogenesis characterized by erratic vascularization (11). It has been shown that autocrine and paracrine signaling by ET-1 modulates cell proliferation, apoptosis, migration, epithelial-to-mesenchymal transition, chemoresistance and neovascularization. In our past studies, we found that glycemic control with metformin was also associated with reduced levels of plasma ET-1 and vascular ETA receptors (12). Building upon this finding and published reports on the role of ET-1 in tumor angiogenesis, we next investigated the role of ET-1 in the dysfunctional angiogenesis in the Goto-Kakizaki (GK) model of type 2 diabetes and showed that bosentan prevents and reverses pathological cerebral neovascularization (9). These findings raised the possibility that modulation of the ET system may offer a therapeutic strategy in diabetes-mediated vascular disease.

DPP-4 inhibitors are a relatively new class of oral glucose-lowering agents that regulate blood glucose. DDP-4 is a ubiquitous enzyme found in many tissues and glucagon-like peptide 1 (GLP-1) is one of its wide spectrum of substrates (13). DPP-4 inhibitors, known as gliptins, prevent the degradation of GLP-1 and thereby promote insulin secretion while reducing glucagon levels. However, there is accumulating evidence that these agents have anti-inflammatory and vasoactive properties independent of glucose-lowering actions (13, 14). The effect(s) of DPP-4 inhibitors on pathological neovascularization and equally important on the ET system was unknown. Retina is another microvascular target of diabetes that presents with pathological neovascularization. Retinal acellular capillary formation is a surrogate marker for pathological retinal ischemia and retinopathy in diabetes (15). Development of acellular capillaries is a hallmark of microvascular degeneration with endothelial cell death leaving only vascular basement membrane. Accordingly, the current study aimed to determine the impact of linagliptin on dysfunctional cerebral and retinal angiogenesis in diabetes in vivo and also investigate the interaction with the ET system using an in vitro brain microvascular endothelial cell culture model.

Methods

Animals

All experiments were performed using male Wistar rats from Harlan (Indianapolis, IN), diabetic GK rats (In-house bred, derived from the Tampa colony or purchased from the Tampa colony, Taconic; Hudson, NY). The animals were housed at the Georgia Regents University animal care facility that is approved by the American Association for Accreditation of Laboratory Animal Care. All protocols were approved by the institutional animal care and use committee. Animals were fed standard rat chow and tap water ad libitum. Body weights and blood glucose measurements were taken biweekly. Blood glucose (BG) measurements were taken from tail vein samples using a commercially available glucometer (Freestyle, Abbott Diabetes Care, Inc; Alameda, CA).

Animal treatments

Treatment started at 24 weeks of age after the development of diabetes-induced cerebrovascular remodeling and neovascularization for 4 weeks. Linagliptin was added to reconstituted rat chow at a concentration of 166mg/kg chow.

Assessment of neovascularization parameters

Vascularization patterns and density were measured using the space-filling fluorescein isothiocyanate (FITC)-dextran method as we recently described (10, 16). Brains were fixed, sectioned and mounted on slides. Z-stacked confocal images of the regions proximate to the middle cerebral artery (MCA) and its branches that supply the frontal motor cortex, bregma 1 to −1 were acquired using Zeiss LSM 510 upright confocal microscope. A mean of 3 values from this region was recorded as an observation. Each measurement from one animal was comprised of an average of 6 images from either the cortical or striatal region. Morphometry was assessed using Fiji and Volocity 6 software.

Acellular capillary measurement

Enucleated eyes were fixed with 1% paraformaldehyde overnight. Dissected retina cups were washed in phosphate-buffered saline, and then incubated with 3% Difco-Trypsin 250 (BD Biosciences, San Jose, CA) in 25 mM Tris buffer, pH 8, at 37 °C for 1.5–2 hours. Eye vasculature was then washed with 5% Triton X-100 to get rid of the neural retina. The transparent vasculature was stained with periodic acid-Schiff and hematoxylin (PASH). Retinal vasculature images were acquired using an Axiovert 200 microscope (Carl Zeiss MicroImaging, Thornwood, NY). Acellular capillaries were identified as capillary-sized blood vessel tubes having no nuclei anywhere along their length. The number of acellular capillaries were counted in eight different fields of the mid-retina and then averaged together, indicating the number/high power field of each image.

Cell culture

BMVECs from Wistar or GK rats were isolated as described previously (10, 16). Cells were grown in MCDB131 medium (Gibco BRL). Experiments were performed using cells between passages 4 and 6. Cells were switched to serum-free medium 6 hrs. before cell migration assay or treatment application. Cells were treated with linagliptin at a concentration of (100 nM). For co-treatment studies, bosentan (10 µM) was added to cells 15 minutes before addition of linagliptin.

Cell migration

The wound-healing assay was performed as described previously (10, 16). Briefly, BMVECs were grown to form a monolayer that is scratched. Wounds are imaged at zero time and at after 24 hours. Percent migration was calculated and blotted as percent to control.

Tube formation

Tube formation assay was performed using growth factor-reduced Matrigel (BD Biosciences) as described previously (10, 16). Briefly, BMVECs were counted and plated at 2·104 cells/ml with Matrigel in a 96 well-plate. Eighteen hours later, images of the tube-like structures were captured using an Axiovert 200 microscope (Carl Zeiss MicroImaging, Thornwood, NY) and analyzed using image J software. Results were represented as percentage to control.

Quantitative real-time PCR

The One-Step qRT-PCR kit (Invitrogen) was used to amplify 10 ng cellular mRNA and quantification was performed as described previously (9). PCR primers to amplify preproendothelin-1 (PPET-1), endothelin B receptor (ETB) and 18S were purchased from Integrated DNA Technologies, Inc. EDN-1 primers: F: 5′-GACTTTCCAAGGAGCTCCAGAA-3′, R: 5′-CAGCTCCGGTGCTGAGTTC-3′. ETB primers: F: 5′-GCTAGGCATCATCGGGAACTC-3′, R: 5′-TTGCGCATGCACTTGTTCTT-3′. Quantitative PCR was performed using a Realplex Master cycler. Expression of PPET-1, ETB was normalized to the 18S level and expressed as relative expression to control.

Endothelin-1 chemiluminescent immunoassay

ET-1 levels in the cell culture supernatant of BMVECs from diabetic animals or cells treated with linagliptin were assessed using the QuantiGlo Endothelin-1 Chemiluminescent Immunoassay (Bioteck, R&D, USA) according manufacturer’s protocol and reported as % of the ET-1 levels in control untreated cells.

Western blots

Equal protein loads of cellular lysate in RIPA buffer (Millipore, Billerica, MA) were separated on a 10% SDS-polyacrylamide gel by electrophoresis. Rat specific anti-ETB receptor and anti-β-actin antibodies were purchased from (Abcam, Cambridge, MA, USA). Primary antibodies were detected using a horseradish peroxidase–conjugated antibody and enhanced chemiluminescence (GE Healthcare, Piscataway, NJ). Relative optical densities of immunoreactivity were determined by densitometry software (Alpha Innotech, Protein Simple, San Jose, CA).

Statistical analysis

Two-way ANOVA was used to assess disease and treatment effects. A Bonferroni’s post-test adjustment for multiple comparisons was used for all post-hoc mean comparisons for significant effects from all analyses. Data was expressed as Mean ± SEM and p<0.05 was considered significant.

Results

Effect of linagliptin on established cerebral neovascularization in diabetes

Diabetic rats showed significant increase in vascular density, vascular volume as well as surface area in both cortex and striatum regions. Linagliptin treatment did not lower blood glucose in diabetic rats (240 ± 25 vs 193 ± 40 mg/dl at the beginning and end of linagliptin treatment, respectively). However, treatment reversed diabetes-induced neovascularization as demonstrated by significant decreased vascular volume, vascular density and surface area as compared to untreated diabetic rats (Fig. 1A–D).

Fig. 1. Linagliptin decreases pathological neovascularization indices in diabetes.

Fig. 1

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24 week old nondiabetic Wistar (C) and diabetic GK rats (D) were treated for 4 weeks with linagliptin 166 mg/kg chow (C+Lina, D+Lina). At termination, rats were injected FITC-dextran to fill and visualize blood vessels. Images acquired by confocal microscopy were used for 3-dimensional reconstruction of the cerebrovascular network for measurement of vascular density, vascular volume, and surface area in the cortex and striatum regions using Volocity 6 software. Representative images from cortex (upper panel) and striatum (lower panel) are shown on Panel (A) and average data are shown in histograms on Panels (B–D). Linagliptin treatment significantly reduced vascular volume, vascular density and surface area as compared to vehicle-treated diabetic GK rats (Results are expressed as mean ± SEM, n=3–5, *p<0.05 vs C, #p<0.05 vs D).

Effect of linagliptin on established small vessels remodeling in diabetes

Similar to neovascularization indices, branch density and tortuosity were significantly higher in diabetic rats compared to controls (Fig. 2A–C). Treatment with linagliptin showed significant reduction in branching when compared to untreated diabetic rats (Fig. 2A–C).

Fig. 2. Linagliptin decreases small vessels remodeling in diabetes.

Fig. 2

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Confocal images used for vascular density measurements in Fig. 1 were converted to binary mode and skeletonized using Image J software to determine branch density and tortuosity in the cortex area. (A) Representative binary (top panel) and skeleton images (bottom panels) of brain sections of Wistar and GK rats treated with vehicle or linagliptin. Linagliptin treatment significantly reduced tortuosity (B) and branch density (C) compared with vehicle-treated GK rats (Results are expressed as mean ± SEM, n=3–5, *p<0.05 vs C, #p<0.05 vs D)

Effect of linagliptin on acellular capillaries in diabetes

Our results showed that diabetic GK rats have significant increase in the number of cellular capillaries compared to control rats. Treatment with linagliptin significantly decreased retinal acellular capillaries formation (Fig. 3A–B)

Fig. 3. Linagliptin decreases retinal acellular capillaries in diabetes.

Fig. 3

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Retinal acellular capillary formation is a surrogate marker for pathological neovascularization in diabetes. 24 week old nondiabetic Wistar (C) and diabetic GK rats (D) were treated for 4 weeks with linagliptin 166mg/kg chow (C+Lina, D+Lina) and capillary-like structures that contain only basement membrane were counted (arrows) (n=4–5, *p<0.05 vs C, #p<0.05 vs D).

Effect of linagliptin on endothelial angiogenic properties in diabetes

BMVECs isolated from diabetic GK rats experienced augmented migration and tube formation. Treatment with linagliptin significantly reduced augmented angiogenic properties in these cells (Fig. 4A–B). While treatment with bosentan alone did not impact the migratory properties of BMVECs from diabetic animals, it negated the inhibitory effect of linagliptin on EC migration (Fig. 4C).

Fig. 4. Linagliptin restores augmented endothelial cell angiogenic properties in diabetes.

Fig. 4

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BMVECs from diabetic animals (D) showed augmented angiogenic signaling as shown by increased tube formation compared to endothelial cells from Wistar control group (C). (A) Treatment with Linagliptin (100 nM) significantly decreased endothelial cell migration of GK rats (n=3–4, *p<0.05 vs C, #p<0.05 vs D). (B) In parallel, treatment with Linagliptin (100 nM) significantly decreased tube formation of GK BMVECs (n=3–4, *p<0.05 vs C, #p<0.05 vs D). (C) BMVECs from diabetic animals treated with vehicle (D), Linagliptin (100 nM) (D+Lina), Bosentan (10 µM) (Bos) or both linagliptin and bosentan (Bosentan was applied 15 min before linagliptin) (D+Lina+Bos). While treatment with bosentan alone did not impact the migratory properties of BMVECs from diabetic animals, it negated the inhibitory effect of linagliptin. (n=3, *p<0.05 vs D).

Effect of linagliptin on the ET system in diabetes

Cells from diabetic rats showed elevated ET-1 production at mRNA and protein levels as compared to cells obtained from control rats (Fig. 5A and B). Interestingly, linagliptin treatment significantly increased PPET-1 expression in both control and diabetic GK cells. Linagliptin treatment did not change ET-1 levels in control cells while decreasing ET-1 in cells from diabetic animals. On the other hand, BMVECs isolated from diabetic GK rats expressed less ETB receptors. Treatment with linagliptin significantly increased ETB receptor transcription and expression in BMVEC isolated from diabetic GK rats (Fig. 5C and D).

Fig. 5. Linagliptin modulates the ET system in diabetes.

Fig. 5

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(A) PPET-1 transcription was assessed in control (C) and diabetic (D) BMVECs. Treatment with linagliptin showed significant increase in the PPET-1 mRNA levels (n=3, *p<0.05 vs C). (B) While BMVECs from diabetic animals showed 4-fold higher levels of ET-1 in the media, treatment with linagliptin reduced ET-1 levels. (n=3, *p<0.05 vs C, #p<0.05 vs D). (C–D) BMVECs from diabetic animals showed lower ETB mRNA and protein levels as compared to control cells. Treatment of control (C+Lina) and diabetic (D+Lina) BMVECs with linagliptin showed significant increase in the ETB mRNA and protein levels (n=3, *p<0.05 vs C, #p<0.05 vs GK).

Discussion

The current study presents novel information that 1) diabetes-induced pathological cerebral neovascularization can be reversed using DPP-4 inhibitor linagliptin in vivo, 2) linagliptin effects are independent of glycemic control, 3) linagliptin reduces ET-1 levels while increasing ETB receptors in brain microvascular endothelial cells, and 4) linagliptin-mediated decrease in proangiogenic properties of endothelial cells is reversed by ETB receptor antagonism with bosentan.

While diabetes promotes impaired angiogenic response and reduced collateral formation in the peripheral circulation, in the eye there is increased yet pathological neovascularization resulting in diabetic retinopathy. We have recently expanded our knowledge of the cerebrovascular architecture in diabetes and demonstrated that there is dysfunctional neovascularization in the cerebrovasculature of diabetic GK rats. We reported increased vascular density, volume, and surface area in the brain parenchyma that was associated with poor vessel wall maturity as indicated by reduced pericytes and increased non-perfused vessels and permeability (5). We also showed similar changes in yet another model of diabetes, db/db mice, suggesting that this pathological neovascularization is not unique to the GK model of diabetes and may have a broader impact in diabetes (5). Equally important our studies showed that glycemic control with metformin either when started at the onset of diabetes or after established microvascular disease, was associated with improvement of angiogenic parameters. Large clinical trials demonstrated that glycemic control is associated with improvement of microvascular complications and we attributed the prevention or reversal of this pathological neovascularization with metformin to its blood glucose lowering effects. Metformin activates the AMP-activated protein kinase (AMPK), a master energy regulator, and also exerts direct antioxidant effects raising the possibility that metformin effects may be independent of its blood glucose lowering effects (10). Thus, in the current study we used linagliptin, a DPP-4 inhibitor, to investigate whether this protective effect is found in other agents commonly used for the treatment of type 2 diabetes. Our findings demonstrate that linagliptin when started late in the disease after microangiopathy is established can reverse pathological angiogenesis as evidenced by decreased vascular volume, density and branching. Furthermore, formation, a hallmark of pathological angiogenesis in diabetic retinopathy (15), was also attenuated. Interestingly these effects were independent of changes in blood glucose as linagliptin did not lower blood glucose in our model. DPP-4 inhibitor class of oral glucose-lowering agents regulates blood glucose by increasing the availability of GLP-1 which then promotes insulin secretion. Since the GK model of diabetes presents with insulin resistance, linagliptin was not able to provide glycemic control in our model, yet provided cerebrovascular protection by reversing pathological neovascularization.

Given that DPP-4 inhibitors display pleiotropic effects independent of glycemic control (13, 14) and our previous study showed that inhibition of the ET system by bosentan also prevented and reversed dysfunctional cerebral angiogenesis in the GK model of diabetes (17), we next investigated the direct effects and interaction of linagliptin with ET-1 on proangiogenic properties of primary BMVECs isolated from control or diabetic animals. As we previously reported (16), BMVECs from diabetic GK rats have erratic migratory properties that resemble the chaotic vascularization seen in vivo and our current data show that linagliptin decreases tube formation and migration in these cells. Interestingly, GK cells also synthesize greater levels of ET-1 than observed in control cells and linagliptin reduces mature ET-1 levels. Intriguingly, linagliptin increases PPET-1 mRNA in both control and diabetic GK cells while this is not the case in mature ET-1 production suggesting that either translation of mRNA and/or processing of the PPET-1 to ET-1 is affected by linagliptin and remains to be determined. Another interesting finding was that cells from diabetic animals have less ETB receptors both at the mRNA and protein level. Linagliptin stimulated ETB mRNA and protein levels both in control and diabetic GK cells. However, whether this increase in ETB receptor contributes to improvement of migratory properties is less clear because when cells are treated with bosentan alone there is no effect on migration. On the other hand, when cells are treated with linagliptin and bosentan, improvement of migratory properties observed with linagliptin alone is lost suggesting that ETB signaling does not directly regulate the endothelial migration but interferes with pathways involved in linagliptin-mediated protection.

DPP-4 is a ubiquitously expressed enzyme and it is found in endothelial cells. In addition to GLP-1, it has multiple substrates as recently reviewed (13). An earlier study reported that DPP-4 mediates endothelial cell migration by cleaving neuropeptide Y (NPY), a sympathetic neurotransmitter with angiogenic properties (18). Another study reported that inflammatory cytokine-mediated microvascular endothelial growth can be regulated by DPP-4 inhibition (19). Collectively, these suggest that DPP-4 can act on one or more angiogenic factors produced by endothelial cells to regulate migration and tubulogenesis. However, the identity of this angiogenic factor in our study remains to be determined.

There are several shortcomings of this study that need to be recognized. In our in vivo work, we were not able to assess cerebrovascular ET-1 levels due to special processing of brains for neovascularization studies. We do not know how linagliptin affected tissue ET-1 levels and whether this correlated with linagliptin-mediated prevention of pathological angiogenesis. In cell culture studies, linagliptin modestly reduced ET-1 levels (~25%) and this was associated with roughly 50% improvement of the migratory properties. Second, our in vitro studies suggest that ETB blockade alone does not affect migration but in our previous work showed that bosentan was effective in preventing and reversing dysfunctional neovascularization. This may have stemmed from short-term nature of cell culture studies and/or the influence other cell types on angiogenesis in the in vivo setting. Third, Patel et al showed that an ETA receptor antagonist directly applied to the eye prevents pathological angiogenesis in oxygen-induced retinopathy. In the current study we used BMVECs that express only the ETB receptor and hence did not include an ETA selective antagonist. Comparison of selective versus nonselective ET receptor antagonism on pathological angiogenesis in vivo remains to be determined. Nevertheless we conclude that 1) linagliptin is effective in reversing established pathological neovascularization and offers therapeutic potential; 2) ET system contributes to pathological neovascularization in diabetes as evidenced by restoration of functional angiogenesis by bosentan treatment and prevention of linagliptin mediated improvement of angiogenesis in the in vitro model; and 3) Effect of linagliptin on regulation of the endothelial ET system requires further investigation since this study investigated short term effects in endothelial cell culture.

Acknowledgements

Adviye Ergul is a Research Career Scientist at the Charlie Norwood Veterans Affairs Medical Center in Augusta, Georgia. This work was supported in part by VA Merit Award (BX000347), VA Research Career Scientist Award, NIH award (NS070239, R01NS083559) and a research grant from Boehringer Ingelheim Pharmaceuticals, Inc. to Adviye Ergul; and American Heart Association Postdoctoral Fellowship (14POST19580004) to Mohammed Abdelsaid. The authors meet criteria for authorship as recommended by the International Committee of Medical Journal Editors (ICJME) and were fully responsible for all aspects of the trial and publication development. The contents do not represent the views of the Department of Veterans Affairs or the United States Government.

Footnotes

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Conflict of interest statement

Authors declare no conflict of interest.

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