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.2023 Feb 15;16(1):25.
doi: 10.1186/s13041-023-01013-1.

Contraction of human brain vascular pericytes in response to islet amyloid polypeptide is reversed by pramlintide

Affiliations

Contraction of human brain vascular pericytes in response to islet amyloid polypeptide is reversed by pramlintide

Cristina Nuñez-Diaz et al. Mol Brain..

Abstract

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 relaxation 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 microvasculature, 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; Blood flow; Diabetes; Mural cells; Vasculopathy.

© 2023. The Author(s).

PubMed Disclaimer

Conflict of interest statement

Malin Wennström has acquired research support (for the institution) from Eli Lilly.

Figures

Fig. 1
Fig. 1
Effects 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
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
Fig. 3
Fig. 3
Effects 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
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
Fig. 5
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
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