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The islet amyloid polypeptide (IAPP), a pancreas‑produced peptide, has beneficial functions in its monomeric form. However, IAPP aggregates, related to type 2 diabetes mellitus ( T2DM), are toxic not only for the pancreas, but also for the brain. In the latter, IAPP is often found in vessels, where it is highly toxic for pericytes, mural cells that have contractile properties and regulate capillary blood flow. In the current study, we use a microvasculature model, where human brain vascular pericytes (HBVP) are co‑ cultured together with human cerebral microvascular endothelial cells, to demonstrate that IAPP oligomers (oIAPP) alter the morphology and contractility of HBVP. Contraction and relaxa‑ tion of HBVP was verified using the vasoconstrictor sphingosine ‑1‑phosphate (S1P) and vasodilator Y27632, where the former increased, and the latter decreased, the number of HBVP with round morphology. Increased number of round HBVP was also seen after oIAPP stimulation, and the effect was reverted by the IAPP analogue pramlintide, Y27632, and the myosin inhibitor blebbistatin. Inhibition of the IAPP receptor with the antagonist AC187 only reverted IAPP effects partially. Finally, we demonstrate by immunostaining of human brain tissue against laminin that individuals with high amount of brain IAPP levels show significantly lower capillary diameter and altered mural cell morphology compared to individuals with low brain IAPP levels. These results indicate that HBVP, in an in vitro model of microvas‑ culature, respond morphologically to vasoconstrictors, dilators, and myosin inhibitors. They also suggest that oIAPP induces contraction of these mural cells and that pramlintide can reverse such contraction. Keywords Amylin, Vasculopathy, Mural cells, Diabetes, Blood flow Introduction The islet amyloid polypeptide (IAPP), also known as amylin, is a 37 aa polypeptide expressed by pancre- atic islet β-cells, where it is postprandially co-secreted *Correspondence: with insulin. In its monomeric form, IAPP physiological Malin Wennström functions include delaying gastric emptying, promoting firstname.lastname@example.org 1 satiation, and reducing postprandial glucagon secretion Cognitive Disorder Research Unit, Department of Clinical Sciences Malmö, Lund University, Malmö, Sweden . However, oligomeric and fibrillar forms of IAPP are Netherlands Institute for Neuroscience, Meibergdreef 47, 1105 related with pathological conditions, such as type 2 dia- BA Amsterdam, The Netherlands 3 betes mellitus (T2DM). This disease is characterized by Faculty of Medicine, Department of Clinical Sciences, Lund, Mass Spectrometry, Lund University, Lund, Sweden the accumulation of IAPP aggregates in the pancreas [2– Department of Experimental Medical Science, Lund University, Lund, 4]. Besides, the polypeptide has been linked with Alzhei- Sweden mer’s disease (AD), since IAPP deposits have been found © The Author(s) 2023. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/. The Creative Commons Public Domain Dedication waiver (http:// creat iveco mmons. org/ publi cdoma in/ zero/1. 0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Nuñez‑Diaz et al. Molecular Brain (2023) 16:25 Page 2 of 11 in the brains of AD patients, regardless of T2DM diag- cell culture medium (PM, ScienCell, #1201), respectively. nosis. The pancreatic IAPP is known to cross the blood For monolayer culture, endothelial cells were grown brain barrier (BBB) [5, 6] and under normal conditions in rat tail collagen I (Gibco, #A1048301) coated 6-well bind to amylin receptor in specific brain regions and plates and HBVP were cultured in poly-L-lysine (Scien- thereby regulate appetite . However, in AD patients Cell #0413) coated 6-well plates, both in humidified air the peptide is found as deposits in the parenchyma, ves- with 5% CO at 37 °C until 70–80% confluent. sel walls, and perivascular space, often co-localizing with amyloid beta (Aβ) [7, 8], which is one of the main Proteomics pathological hallmarks of AD. This is interesting given The HBVP were evaluated prior to experiment using pro - that the risk of AD in T2DM patients is strongly linked teomics. The HBVP were grown in poly-L-lysine (Sci - to vascular complications (e.g., microvascular disease, enCell #0413) coated 12-well plates in 3 replicates and diabetic foot, cerebrovascular disease, cardiovascular thereafter lysed in RIPA buffer (Sigma Aldrich #R0278). disease) associated with the disease . The significance For proteomics, 100 µL was reduced with dithiotreitol of vessel-associated IAPP depositions in the brain is not and alkylated with iodoacetamide followed by protein known, but a previous study has shown that IAPP accu- precipitation with ice cold ethanol overnight at − 20 °C. mulation in brain vessel leads to loss of thight junctions The samples were centrifuged and the pellets were resus - and decreased endothelial cell coverage in rats . In pended in 100 mM ammonium bicarbonate and protein addition, we have demonstrated that oligomeric IAPP concenteration were determined. Samples, 30 µg, were (oIAPP), but also IAPP fibrils (although to a lesser extent digested with trypsin overnight. The generated pep - compared to oIAPP), are highly toxic to cultured primary tides were injected into liquid chromatography–tandem human brain vascular pericytes (HBVP) . Pericytes mass spectrometry (LC–MS/MS). The generated MS/ are mural cells which enclose the capillaries in the brain MS spectra were searched using Proteome Discoverer and form, together with endothelial cells and astrocytes, 2.5 (Thermo Fisher Scientific) against UniProt Human the BBB. Pericytes are therefore implicated in BBB per- (UP000005640). The precursor tolerance and fragment meability, regulation and clearance of debris and toxic tolerance were set to 15 ppm and 0.05 Da, respectively. substances (e.g., Aβ). Pericytes also play a crucial role in Trypsin was selected as enzyme, methionine oxidation angiogenesis and leukocyte extravasation , and regu- and deamidation of aspargine and glutamine were treated late capillary blood flow by relaxing and contracting their as dynamic modification, and carbamidomethylation of endothelial tube enwrapping projections . It may cysteine as a fixed modification. The dataset showed that thus be that IAPP, via its impact on pericytes, strongly the HBVP expressed mural cell markers such as PDGFβ affects Aβ clearance, BBB permeability, and contrac - receptor and NG2, but not PDGFα receptor nor Dcn. tion. Indeed, studies have shown that monomeric IAPP Proteins involved in the contractility machinery, includ- and pramlintide (its non-aggregative analog) enhance Aβ ing transgelin, myosin regulatory light chain 9, tropomy- clearance from the brain to the blood , and an asso- osin beta chain, calponin 1, myosin 9 and myosin 10 were ciation between BBB permeability and total IAPP lev- also found. els in cerebrospinal fluid (CSF) has been reported . Whether IAPP interferes with capillary contraction is Stimuli for cell culture less explored. Therefore, the purpose of this study is to IAPP oligomers were prepared based on a previ- use an in vitro brain microvasculature model to eluci- ously published protocol , which yields IAPP bands date whether human oIAPP affects the morphology and between 4 and 25 kDa on a western blot . Briefly, lyo - contractility of mural cells. We will also investigate the philized synthetic human I APP (AlexoTech AB #AI- 1-37 potential link between IAPP and capillary contraction by 452-10) was solubilized in 10 mM NaOH (pH 11). For analyzing capillary diameters and alterations in mural cell adjusting the pH to 7, phosphate buffer was added to a morphology in the hippocampal region CA1 of individu- final concentration of 100 µM. For oligomer formation, als with high and low amount of IAPP in hippocampus. IAPP was incubated with agitation for 20 min at room temperature. Pramlintide acetate salt (Pram) (Sigma Materials and methods Aldrich #SML2523) Y27632 (Sigma Aldrich, #SCM075), Cells and AC187 (Tocris, #3419) were resuspended in bi- Human cerebral microvascular endothelial cells distilled H O. Sphingosine 1 phosphate (S1P) (Sigma (hCMEC/D3, Millipore, #SCC066) and primary human Aldrich, #73914-1MG) was solubilized in NaOH 0.3 M brain vascular pericytes (HBVP, ScienCell, #1200) were to a stock concentration of 1 mM. Blebbistatin (Blebb) grown in complete endothelial cell growth medium (Tocris, #1760) was resuspended in dimethyl sulfoxide to (EGM-2MV, Lonza, #CC-3129) and complete pericyte a concentration of 100 mM. Nuñez‑Diaz et al. Molecular Brain (2023) 16:25 Page 3 of 11 Matrigel model Individuals included in the study For the Matrigel model, glass bottom angiogen- Human hippocampal samples were provided by the esis µ-slides (Ibidi #81507) were coated with a layer Netherlands Brain Bank (NBB), where neuropathologi- of Matrigel basement membrane matrix (10 mg/mL) cal assessment of Aβ, neurofibrillary tangles and neuritic (Corning # 354234) and incubated for 30 min at 37 °C plaques were performed according to ABC staging . for polymerization. The cells in monolayer culture were NBB also evaluated Lewy bodies (LB) stage according to 70–80% confluent, in passages 4–6 (HBVP) and 31–33 Braak et al. . Sex, age, neuropathological assessment (hCMEC/D3), and with a doubling time of approximately (ABC, LB), IAPP levels, diagnosis of T2DM, postmortem 48 h. HBVP were labelled with CellTracker Green delay, and cause of death of the individuals are shown in CMFDA dye (Invitrogen, #C7025) prior to trypsiniza- Table 1. The individuals included in the study showed lit - tion, following the manufacturer’s instructions. After- tle or no Aβ deposition in hippocampus to avoid inter- wards, hCMEC/D3 and HBVP were co-seeded on top ference of this peptide with vessel diameter. The levels of of the Matrigel layer at a density of 200,000 and 100,000 IAPP in the hippocampus of the individuals were meas- cells/mL, respectively, using 50 µL of medium (EGM- ured previously and described by Schultz et al. . The 2MV 10% FBS) per well. Three hours after seeding, when individuals were divided in two groups based on the the cells have already formed capillary-like structures mean value (10 pg/mL) of their total IAPP levels (solu- (CLS), stimulation was performed with IAPP 10 µM, ble and insoluble), (n = 4) cases with IAPP levels below sphingosine 1 phosphate 0.1 µM, Y27632 1 µM (alone the mean value (IAPP low) (4.83–6.05 pg/mL) and (n = 4) or combined with IAPP), AC187 10 µM (combined with cases with IAPP levels above the mean value (IAPP IAPP), pramlintide 1 µM (alone or combined with IAPP), high) (13.63–15.93 pg/mL). In all cases, informed con- blebbistatin 1 µM (combined with IAPP), and the corre- sent for using brain tissue and clinical data for research sponding vehicle conditions. The model was stimulated was obtained from the patients or their closest relatives with the different conditions for 12 h. After stimulation, in accordance with the International Declaration of Hel- the microvascular model was stained with trypan blue sinki and the Code of Conduct for Brain Banking. The (Bio-Rad, #1450013) for cell death analysis. After 12 h of medical ethics committee of VU Amsterdam approved stimulation, two randomly chosen fields of view of each the tissue collection procedures and the Swedish Ethical well were captured with an Olympus BX41 light micro- Review Authority approved the study. All data were ana- scope with the 10 × objective (6 replicates per condition, lyzed anonymously. 2 fields per replicate). To avoid biases, only after the fields were selected in the bright field microscopy, the images Immunostaining and vessel quantification of fluorescent pericytes were captured. The numbers of The hippocampal tissue was fixed in 4% paraformalde - total HBVP and round HBVP per arm were quantified hyde for 4 h and then cryoprotected in phosphate-buff - in the fluorescent images using ImageJ software, where ered saline with 30% sucrose until it sank. The tissue was a threshold was set for the detection of green labeled sliced into sections of 40 µm thickness using a microtome HBVP, as well as a circularity filter of 0.8–1.0 (where 1.0 (Leica SM 2010R). The sections were stored free-floating is a perfect circle). in cryoprotectant antifreeze solution at − 20 °C. In order to analyze the diameter of brain vessels and mural cell Table 1 Clinical data of individuals included in the study 1 2 Sex Age (years) ABC staging LB (0–6) T2DM IAPP levels Postmortem Cause of death (pg/ml) delay (h:min) M 70 A0B1C0 3 No 4.83 6:20 Pneumonia + cardiogenic shock M 63 A0B1C0 0 Yes 5.18 5:00 Unclear, possible infection M 75 A2B1C1* 0 No 5.70 7:10 Cardiac arrest with COPD F 60 A0B0C0 0 No 6.05 8:10 Breast cancer with metastasis F 92 A0B2C0 1 No 13.63 6:35 Heart failure F 68 A0B0C0 0 Yes 13.67 4:30 Euthanasia M 102 A1B2C1* 0 No 14.54 5:00 Ileus F 69 A0B0C0 0 Yes 15.93 5:25 Cachexia and infection 1 2 ABC Montine staging of Alzheimer’s disease. A0‑3: stain for Aβ/amyloid plaques. B0‑3: stain for neurofibrillary tangles. C0‑3: stain for neuritic plaques. LB: 0–6 Lewy Body staging according to Braak. T2DM: Type 2 Diabetes Mellitus. COPD: chronic obstructive pulmonary disease *0–2 diffuse amyloid plaques found in CA1 Nuñez‑Diaz et al. Molecular Brain (2023) 16:25 Page 4 of 11 morphology, the sections were stained against laminin (Fig. 1C–E). To exclude that these morphological changes α5, expressed by multiple mural cell types and vascular were due to apoptosis/necrosis, we stained with trypan cells, including pericytes and endothelial cells. First, the blue, which specifically labels dead cells (Fig. 1F, G). We sections were incubated in quenching solution (3% H O , detected a very low proportion of trypan blue-positive 2 2 10% methanol) for 30 min, followed by Impress reagent round HBVP (less than 10%), and there were no signifi - kit blocking solution (Vector Laboratories #MP-7402) for cant differences between stimulations with oIAPP and 1 h at room temperature and then incubation with mouse vehicle (Fig. 1H). anti-laminin (clone 4C7, Dako) in blocking solution over- night at 4 °C. Afterwards, the sections were incubated in Validation of the microvascular model Ig Impress reagent kit secondary anti-mouse antibody Since the oIAPP-induced changes in HBVP could not be (Vector Laboratories #MP-7402) at room temperature for explained by cell death, we investigated whether a round 2 h, and then developed for 2 min in 0.25 mg/mL diamin- HBVP morphology is associated with contractile proper- obenzidine and 0.012% H O . The sections were mounted 2 2 ties. Therefore, we analyzed the morphology after stimu - with DPX (Sigma Aldrich, #06522) and pictures of CA1 lating with the vasoconstrictor sphingosine 1-phosphate hippocampal region (stratum lacunosum-moleculare) [22, 23], and the ROCK inhibitor Y27632, a vasodilator were captured with an Olympus BX41 light microscope . Compared with the vehicle condition (Fig. 2A), the with 20 × objective (two sections of each individual, 20 stimulation with S1P (Fig. 2B) resulted in a significantly fields in total). The diameter of total laminin-enclosed higher proportion of round HBVP, whereas after the capillaries and the diameter of laminin-enclosed capillar- treatment with Y27632 (Fig. 2D) this proportion was sig- ies near mural cells were measured by a blinded observer nificantly lower (Fig. 2E, F ). using ImageJ software. Differences in laminin-enclosed mural cell morphology were analyzed by measuring the height of the laminin-enclosed mural cell bodies (dis- Stimulation with agonists and an antagonist of oIAPP tance between the highest point of mural cells and the Next, we aimed to investigate whether the impact of capillary) found in the images using SenseCell software oIAPP on HBVP morphology is mediated via the IAPP (Olympus). More than 70 mural cells per individual were receptor. We challenged the CLS with oIAPP together analyzed. with the IAPP receptor antagonist AC187  and found that the proportion of oIAPP-induced round HBVPs Statistics was no longer significantly higher compared to vehi - Statistical analysis was performed using Prism soft- cle, but no significant difference was detected between ware (version 9.2.0, GraphPad). For normal distribution oIAPP and oIAPP + AC187 treatments (0.359 ± 0.050 vs assessment, Kolmogorov–Smirnov test was performed. 0.277 ± 0.078, p = 0.094) (Fig. 3A–D). We also challenged Our data was normally distributed, therefore, we per- our model with the IAPP receptor agonist pramlintide formed independent-samples t-test or one-way ANOVA but found no alteration in the proportion of round HBVP followed by Dunnett or Tukey test. Results are repre- (Fig. 3E–G). sented as means ± standard deviations. A value of p < 0.05 was considered significant. Stimulation with IAPP combined with Y27632, pramlintide, Results or blebbistatin Analysis of the effects of IAPP stimulation in vitro To investigate the possibility to reverse the impact of As described previously [20, 21], the co-culturing of oIAPP on HBVP morphology, we challenged the micro- endothelial cells and HBVP resulted in self-assembled cell vascular model with oIAPP together with either Y27632, clusters connected by branches, resembling a network pramlintide, or blebbistatin, an inhibitor of muscle and of capillaries without lumen (capillary-like structures, non-muscle myosin II  (Fig. 4A–E). All three mol- CLS) (Fig. 1A, B). The CLS consisted of HBVPs tightly ecules were able to reverse the impact of oIAPP, and associated with the elongated endothelial cells. Many the number of round HBVP was significantly lower in of HBVP were localized in the peripheral part along the these conditions compared with the stimulation with branch (Fig. 1C, D). After stimulating the CLS with oli- only oIAPP (Fig. 4F, G). Supplementation with Y27632 gomeric IAPP (oIAPP) for 12 h, the HBVP displayed a together with oIAPP yielded additionally significantly round shape more frequently (Fig. 1C, D). The number of lower number of round HBVP compared to vehicle. round HBVP per total number of HBVP was significantly higher compared to vehicle condition, which predomi- nantly contained HBVP with an elongated morphology Nuñez‑Diaz et al. Molecular Brain (2023) 16:25 Page 5 of 11 Fig. 1 Eec ff ts of oIAPP on HBVP in the microvascular model. A shows capillary‑like structures (CLS) formed by HBVP and endothelial cells co‑ cultured on a gel matrix. Higher magnification of (A) is seen in (B). Scale bars in (A) and (B): 200 µm. In (C) and (D) CLS after treatment with vehicle (C) and oIAPP (D), with higher proportion of round‑shaped HBVP (black arrows) in the latter. Scale bar in (C) and (D): 100 µm. Quantification of the proportion of round‑shaped HBVP per CLS after stimulation is seen in (E). Images in (F) and (G) show CLS stained with green Cell Tracker (F) and trypan blue (G), where the red arrow indicates a round HBVP negative for trypan blue and the blue arrow indicates a trypan blue‑positive round HBVP. Scale bar in (F) and (G): 50 µm. The quantification of the proportion of trypan blue ‑positive HBVP among total round HBVP per CLS after stimulation is shown in (H). Images (A) and (B) were acquired with the 4 × objective, images in (C), (D), (F) and (G) were acquired with the 10 × objective. Experiments in (E) and (H) were performed in 6 replicates and data was analyzed using student t‑test. Each point represent the mean ± SD. Significant difference at **p < 0.01 Fig. 2 HBVP morphology in the microvascular model after stimulation with controls of contraction. Capillary‑like structures (CLS) (HBVP: green) treated with vehicle (A), with S1P (B), untreated (C), and treated with Y27632 (D) for 12 h, scale bar: 100 µm. Quantification of the proportion of round‑shaped HBVP per CLS after stimulation is seen in (E) and (F). All images were acquired with the 10 × objective. The experiments were performed in 6 replicates and data was analyzed using student t‑test. Each point represent the mean ± SD. Significant difference at *p < 0.05. **p < 0.01 Nuñez‑Diaz et al. Molecular Brain (2023) 16:25 Page 6 of 11 Fig. 3 Eec ff ts of activation/inhibition of IAPP receptor. CLS after stimulation with vehicle (A) , IAPP (B), and IAPP together with the antagonist AC187 (C), scale bar (A–C): 100 µm. Quantification of the proportion of round‑shaped HBVP per CLS after stimulation with vehicle, IAPP, and IAPP with AC187 is seen in (D). Untreated CLS (E) vs. after stimulation with the IAPP agonist pramlintide (F), scale bar in (E) and (F): 100 µm. Graph in (G) shows quantification of the proportion of round‑shaped HBVP per CLS for untreated and pramlintide stimulation. All images were acquired with the 10 × objective. Experiments in (D) and (G) were performed in 6 replicates. In (D), data was analyzed using one‑ way ANOVA followed by Dunnets correction test (n = 2 comparisons), in (G), data was analyzed by student t‑test. Each point represent the mean ± SD. Significant difference at *p < 0.05 Fig. 4 Treatments modifying the effect of oIAPP on HBVP. CLS after stimulation with vehicle (A) , oIAPP alone (B), and oIAPP together with either Y27632 (C), pramlintide (D) or blebbistatin (E), scale bar (A-E): 100 µm. Graphs in (F) and (G) show quantification of the proportion of round‑shaped HBVP per CLS after stimulation. All images were acquired with the 10 × objective. Experiments in (F) and (G) were performed in 6 replicates and data was analyzed using one‑ way ANOVA followed by Tuckey test with (n = 6) comparisons (F) or (n = 3) comparisons (G). Each point represent the mean ± SD. Significant difference at **p < 0.01, ***p < 0.001 Quantification of vessel diameter in human brain tissue the analysis. The diameter of laminin-enclosed capillar - Finally, to investigate the potential association between ies near mural cells was significantly lower in individuals brain IAPP levels and mural cell contraction in clinical with high levels of total IAPP (Fig. 5C). The diameter of material, we analyzed the diameter of laminin-enclosed laminin-enclosed capillaries with no visible laminin- capillaries in the CA1 hippocampal region of neuro- enclosed mural cells was also significantly lower in pathologically evaluated individuals (n = 8). Levels of individuals with high total IAPP levels (Fig. 5D). To inves- total IAPP in homogenates from this region of these indi- tigate whether the mural cell morphology is also affected viduals have been analyzed previously  and the indi- in individuals with high levels of IAPP, we analyzed the viduals were divided into two groups: high (n = 4) and height of the laminin-enclosed mural cell bodies (repre- low (n = 4) IAPP levels. Table 1 shows gender, age, neu- sentative images in Fig. 5E, F). The analysis showed that ropathological assessment (ABC staging and Lewy Bod- individuals with high levels of IAPP showed significantly ies), presence of T2DM, total IAPP levels, postmortem higher height compared to individuals with low levels of delay, and cause of death of the individuals included in IAPP (Fig. 5G). Nuñez‑Diaz et al. Molecular Brain (2023) 16:25 Page 7 of 11 Fig. 5 Analysis of capillary diameter in human tissue. Images in (A) and (B) show immunostainings for laminin in individuals with low (A) and high levels (B) of IAPP. Scale bars: 50 µm (A) and (B), 25 µm (down to the right). Graph in (C) shows quantification of the diameter of laminin enclosed capillaries near laminin enclosed mural cells in individuals with low levels of IAPP (Low IAPP) and high levels of IAPP (High IAPP). Graph in (D) shows quantification of the diameter of laminin enclosed capillaries with no visible mural cells. Images in (E) and (F) show measurement of laminin enclosed mural cells with different heights were image in (E) shows a representative mural cell with low height and (F) represents a mural cell with high height. Scale bar: 5 µm (E) and (F). Black dashed line in (E) and (F) indicate the measured distance. Graph in (G) shows quantification of the height of laminin enclosed mural cells in Low IAPP and High IAPP. Images in (A) and (B) were acquired with the 20 × objective, images in (E) and (F) were acquired with the 40 × objective. Data in (C), (D) and (G) were analyzed using student t‑test. Each point represents the diameter of one capillary in (C) and (D). Each point represents one measurement of mural cell height in (G). Significant difference at ***p < 0.001 altered endothelial calcium wave propagation  and Discussion endothelial cell basal tone and tension  have been The aim of this study was to investigate the effect of found after blebbistatin stimulation. Y27632 has been oIAPP on HBVP morphology and contractility using an shown to suppress endothelial RhoA/ROCK activation in vitro microvascular model. Using the microvascu- and improve endothelial cell viability  and in addi- lar model, we demonstrated that oIAPP cause changes tion increasing the expression of the vascular endothe- in HBVP morphology, displaying a round shape more lial (VE)-cadherin in cell-to-cell junctions . In view frequently. The morphological changes could not be of the known impact of these substances on endothelial explained by increased HBVP death, as the proportion cells, we can only assume that the endothelial cells in our of dead cells was very low compared with the total num- model are also affected in our experiments. Hence, given ber of round HBVP and was not significantly increased the tight reciprocal communication between endothelial by oIAPP stimulation. We, therefore, explored the cells and pericytes , we cannot exclude the possibility impact of oIAPP on HBVP tone. The contractile prop - that the found pericyte reaction (contraction or relaxa- erties of HBVP involve myosin regulatory light chain 9 tion depending on substances) could partly be a result , as well as the RhoA/ROCK pathway . Whether of an indirect pericyte response to endothelial cell reac- oIAPP affects this pathway has not been described tions to the three substances. Nevertheless, our finding before, but Aβ oligomers, which share many proper- suggest that oIAPP, indirectly or directly, and just like Aβ, ties with oIAPP , are known to both activate ROCK induce HBVP contraction, and that this induction might  and to induce pericyte contraction . Hence, to be mediated via the RhoA/ROCK pathway. To date there investigate if HBVP morphological changes were due are no studies (to our knowledge) demonstrating a direct to contraction, we stimulated our model with the vaso- impact of oIAPP in the RhoA/ROCK pathway, but since constrictor sphingosine 1-phosphate, an activator of the oIAPP (just like Aβ oligomers) binds to the receptor for RhoA/ROCK pathway [22, 23], and the ROCK inhibi- advanced glycation end products (RAGE) , and this tor Y27632, a vasodilator , alone and together with receptor is in turn known to activate RhoA/ROCK path- oIAPP. Indeed, S1P alone mimicked the effects of oIAPP way in different cell types (including retinal pericytes on HBVP morphology, and both Y27632 and blebbista- , microvascular pulmonary endothelial cells  tin, a myosin inhibitor , reverted the effect of oIAPP and BV2 microglial cells ), we speculate that oIAPP on HBVP morphology. Of note, all three substances (S1P, induced the contraction in our experiments via RAGE. blebbistation and Y27632) have been shown to affect The impact of IAPP on mural cell contraction in endothelial cells in different ways. S1P promotes for humans has not been reported before, but there are a few example endothelial barrier protection [32, 33] and acti- studies describing an effect of IAPP on the vascular tone vates the endothelial RhoA/ROCK pathway , while Nuñez‑Diaz et al. Molecular Brain (2023) 16:25 Page 8 of 11 in rats. These studies are, however, inconsistent. One isoforms of the receptor (A MY ), formed by com- 1-3(a/b) study indicated that rat IAPP (rIAPP) induced relaxa- binations of CTRa/CTRb with RAMP 1–3, and the dis- tion of rat pulmonary arteries , while another study tribution of the isoforms in different tissues is difficult to revealed reduced relaxation of rat mesenteric arteries assess . Interestingly, our results did not show a clear after rIAPP treatment . Of note, rodent IAPP is non- involvement of the receptor in oIAPP-induced HBVP aggregative, and, hence, corresponds to the monomeric contraction. Although the mean proportion of round and more beneficial version of human IAPP. In addition, HBVP after supplementation with the AMY receptor these experiments were performed in arteries, whose antagonist AC187 together with oIAPP did not signifi - mural cells may respond to IAPP in a different way com - cantly differ from vehicle, it did not either differ from pared to the ones in capillaries. Our study, where we used the proportion of round HBVP after oIAPP stimulation a microvascular model to mimic human capillaries, dem- alone. In addition, stimulation of the receptor with IAPP onstrate that oIAPP induces a key morphological hall- agonist pramlintide had no effect. This could be explained mark of contraction in HBVP. These findings were further by the fact that different isoforms of the receptor have backed-up by our human postmortem study, where we different affinities for oIAPP, AC187, and pramlintide. analyzed the brain capillaries of individuals with high In addition, while it is known that the AMY receptor has and low levels of IAPP. Our results show that individuals an affinity for IAPP in its monomeric/non-aggregative with high levels of total hippocampal IAPP display sig- form, little is known about its affinity for oIAPP. Hence, nificantly smaller laminin-enclosed capillary diameters as it is important to consider other pathways potentially well as a significantly lower number of laminin-enclosed influenced by oIAPP, such as membrane permeabiliza - mural cells with a flattened morphology in CA1. Of note, tion, alternative receptors, or other mechanisms of cell the shift from elongated flat morphology to a more tri - transport. For example, human IAPP (hIAPP) oligomers angular morphology has previously been described as an are known to form pores, which lead to the disruption of indication of pericyte migration . Hence, although a membrane integrity and an increase in reactive oxygen number of studies support the idea that pericyte, in addi- species . hIAPP can also form channels permeable for + + 2+ − tion to smooth muscle cells, have contractile properties Na, K, Ca , and C l in lipid bilayer membranes . (for review see ), we cannot rule out that migration of Whether the latter mechanism also occurs in mural cells pericytes in the analyzed individuals might have affected has not been investigated yet, but if it does, it is tempt- the results. Moreover, the ELISA used for analysis of hip- ing to speculate that this leads to increased intracellular 2+ pocampal IAPP does not distinguish between aggrega- concentration of Ca , which, in turn, would increase tion forms, and hence our results do not reveal whether contraction and eventually lead to apoptosis. Also, other the reduced capillary diameter is related to oIAPP levels receptors beside the AMY receptor may be implicated. or higher amount of IAPP in general (i.e. monomers, oli- For example oIAPP also binds to RAGE [51, 52]. This gomer or fibrils). It should further be emphazised that receptor, which is found on pericytes [53, 54], is known previous studies have shown an impact of Aβ on peri- to mediate the toxic effects induced by IAPP in pancre - cyte contraction , therefore, we selected cases with atic β-cells . Hence, we cannot rule out the possibil- no or very scarce amyloid pathology in the hippocam- ity that RAGE or other receptors with affinity for IAPP pus. However, most elderly individuals display some Aβ are also involved in the oIAPP-induced HBVP morpho- pathology, despite the lack of neurodegenerative changes logical changes shown in this study. Finally, oIAPP might linked to Alzheimer’s dementia or other dementia forms. also be internalized by mural cells via different mecha - Therefore, we cannot completely rule out the possibility nisms of cellular transport and thereby cause morpho- that Aβ pathology, not captured by the neuropathological logical changes. A support for this idea can be found in evaluation (such as Aβ oligomers), also has an impact on studies describing an uptake of oIAPP via translocation, the analyzed capillary diameter. In addition, the number micropinocytosis, and clathrin-mediated endocytosis in of cases included in the study was low (due to shortage pancreatic β-cells . All these possible mechanisms of of cases completely free from Aβ pathology), which is a oIAPP might act alone or combined, provoking the mor- limitation of the study. Nevertheless, taken together, our phological changes we detected in the HBVP/mural cells. findings point towards a direct impact of IAPP on mural Although the AMY receptor antagonist AC187 was cells, inducing capillary contraction in humans. unable to revert oIAPP-induced morphological changes Next, we investigated the role of IAPP receptor in in HBVP, we found that Y27632, blebbistatin, and the morphological changes observed in the HBVP in pramlintide had a potent reversal effect. Both Y27632 our model. The IAPP receptor (AMY) is a heterodimer and blebbistatin are inhibitors of contraction [24, 26], consisting of the calcitonin receptor (CTR) and a recep- which supports the idea that oIAPP induces an increase tor activity-modifying protein (RAMP) . There are 6 in HBVP contraction. Regarding the mechanism of Nuñez‑Diaz et al. Molecular Brain (2023) 16:25 Page 9 of 11 Abbreviations action of pramlintide, it might be explained by a physi- AD Alzheimer’s disease cal interaction with IAPP since previous studies have AMY The IAPP receptor shown that pramlintide inhibits hIAPP aggregation BBB Blood brain barrier Blebb Blebbistatin in vitro . Another possible mechanism could be that CA1 Cornu Ammonis pramlintide competes with oIAPP for the same recep- CLS Capillary‑like structures tors and given its non-aggregative nature, pramlint- COPD Chronic obstructive pulmonary disease CSF Cerebrospinal fluid ide binding elicits a counteracting effect on the HBVP. HBVP P rimary human brain vascular pericytes From this perspective it is noteworthy that pramlintide, hCMEC/D3 Human cer ebral microvascular endothelial cells an FDA approved drug used together with insulin for IAPP Islet amyloid polypeptide LB Lewy bodies T2DM treatment, has well-described beneficial effects. LC–MS/MS Liquid chromatography–tandem mass spectrometry Apart from mimicking the physiological effects of mon - LRP‑1 Low‑ density lipoprotein receptor‑related protein 1 omeric IAPP , pramlintide improves learning and oIAPP Oligomeric IAPP Pram Pramlintide memory function in diabetic rats , and reverts the RAGE Receptor for advanced glycation end products depression of the long-term potentiation caused by Aβ RAMP Receptor activity‑modifying protein and oIAPP in mouse brain slices . Besides, pram- S1P Sphingosine‑1‑phosphate T2DM Type 2 diabetes mellitus lintide enhances the removal of Aβ from the brain over the BBB, increasing the trafficking of the low-density Acknowledgements lipoprotein receptor-related protein 1 (LRP-1), a well- The authors wish to thank NBB for providing the human brain samples, neuropathological evaluation, and clinical data, Prof. Peter Nilsson (Linköping known Aβ clearance transporter, to the membrane of University, Sweden) for the generous donation of the amyloid‑binding dye endothelial cells in the BBB . Since pericytes play HS‑169, Prof. Louise Torp Dalgaard (Roskilde University, Denmark) and Dr. an important role in this LRP-1 mediated Aβ clear- Kim Henriksen (Nordic Bioscience) for intellectual support and the Center for Translational Proteomics at Medical Faculty, Lund University for the proteomic ance , it is tempting to speculate that the beneficial analysis of HBVP. impact of pramlintide on Aβ clearance is in part medi- ated via its impact on pericyte/mural cell functionality Author contributions NBB provided the human brain tissue. MW and CND conceptualized the study as seen in our study. and designed the experiments. CND and NS performed the experiments and Finally, the model used in our study admit investiga- analysis of data. MW provided financial support. CND and MW drafted and tions on HBVP when interacting with endothelial cells, edited the final version of the manuscript. CW performed the protemics. DP, NS and KS gave intellectual support and edited the manuscript. All authors which is different from monolayered in vitro mod - read and approved the final version. els. Such system is useful when investigating isolated events (such as oIAPP exposure), but it is important to Funding Open access funding provided by Lund University. This study was financially point out that cell cultures can never replicate a biolog- supported by the Swedish Research Council (MW ), Brain Foundation (MW ), ical system. Nevertheless, our findings point towards a Olle Engkvists foundation (MW and CND), Dementia Foundation (MW ), Greta contraction effect of oIAPP on pericytes, and that this and Johan Kockska foundation (MW and CND), the Åhlén foundation (MW ). contraction can be reverted by pramlintide and con- Availability of data and materials traction inhibitors. The significance of these findings The dataset supporting the conclusions of this article is available via Pro‑ may be several-fold. First, given that mural cells, like teomeXchange with the identifier PXD035048. pericytes, regulate basal capillary blood flow resist - ance in the brain [61, 62], vascular accumulation of Declarations aggregated IAPP, as seen in demented T2DM patients Ethics approval and consent to participate and patients with AD, may underly the reduced blood Informed consent for using human tissue and clinical data for research was flow in these patients. Secondly, although our studies obtained from the patients or their closest relative in accordance with the International Declaration of Helsinki and the Code of conduct for Brain Bank‑ focus on the impact of IAPP on mural cells in the brain, ing. The medical ethics committee of VU Amsterdam approved the tissue col‑ it is likely that similar cells in the periphery and retina lection procedures and the regional ethical board in Lund approved the study. also respond to IAPP in a similar way, an idea impor- Consent for publication tant to consider in future research on vascular com- Not applicable. plications in T2DM (such as retinopathy and diabetic foot). Thirdly, the ameliorating effect of pramlintide on Competing interests Malin Wennström has acquired research support (for the institution) from Eli oIAPP-induced contraction highlights the potential use Lilly. of pramlintide not only for glucose control, but also to prevent vascular complications. Hence, we conclude Received: 13 December 2022 Accepted: 5 February 2023 that the impact of oIAPP on pericyte contraction might be an event important to target. Nuñez‑Diaz et al. Molecular Brain (2023) 16:25 Page 10 of 11 References 22. Coussin F, Scott RH, Wise A, Nixon GF. 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Pramlintide antagonizes beta amyloid (Aβ)‑ and human amylin‑induced depres‑ Re Read ady y to to submit y submit your our re researc search h ? Choose BMC and benefit fr ? Choose BMC and benefit from om: : sion of hippocampal long‑term potentiation. Mol Neurobiol [Internet]. 2017;54:748–54. fast, convenient online submission 60. Ma Q, Zhao Z, Sagare AP, Wu Y, Wang M, Owens NC, et al. Blood‑brain thorough peer review by experienced researchers in your ﬁeld barrier‑associated pericytes internalize and clear aggregated amyloid‑β42 by LRP1‑ dependent apolipoprotein e isoform‑specific mechanism. Mol rapid publication on acceptance Neurodegener [Internet]. 2018;13. support for research data, including large and complex data types 61. Hartmann DA, Coelho‑Santos V, Shih AY. Pericyte control of blood flow • gold Open Access which fosters wider collaboration and increased citations across microvascular zones in the central nervous system. 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Molecular Brain – Springer Journals
Published: Feb 15, 2023
Keywords: Amylin; Vasculopathy; Mural cells; Diabetes; Blood flow
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