Choline supplementation improves cognitive performance in frail hypertensive patients: novel insights on endothelial function from the INTERVENTIONIST study

Graphical Abstract

Graphical Abstract.

Frailty is a prevailing condition in hypertensive older adults, increasing the risk of cognitive impairment and adverse outcomes.^1–3 Choline supplementation has shown beneficial effects on cognitive impairment.^4,5 However, the exact mechanisms underlying these favorable actions have not been explored. We hypothesized that choline could improve endothelial dysfunction, a well-established hallmark of hypertension and frail aging^6,7 and attenuate endothelial cell senescence.

Thus, we designed a study to test the hypothesis that ‘cholINe alfosceraTE impRoVEs cogNiTIve perfOrmance iN fraIl hypertenSive paTients’ (INTERVENTIONIST), enrolling consecutive frail hypertensive older adults presenting from March 2021 to June 2022 at the Local Health Unit (ASL) of the Italian Ministry of Health of Avellino, Italy. All patients met the following inclusion criteria: age >65 years; confirmed diagnosis of primary hypertension and frailty; global cognitive performance with a Montreal Cognitive Assessment⁶ score <26. The diagnosis of frailty was reached following the Fried Criteria, as described.⁶ Patients were divided into two groups: receiving choline alfoscerate [alpha-glycerylphosphorylcholine (α-GPC)] 1200 mg/day or not (control). The protocol was approved by the Ethical Committee of Campania Nord; all patients (or their legal representatives) signed an informed consent.

For in vitro experiments, the wound healing assay (to evaluate cell migration) and the network-like assay (to quantify angiogenesis) were performed in human umbilical vascular endothelial cells (HUVECs, passages 3–6) as described⁸; images were obtained using Incucyte^® S3 Live-Cell Analysis System (Essen Bioscience, Ann Arbor, MI) and analyzed via the image processing software FIJI (Fiji is justImageJ). The Senescence β-galactosidase staining kit (Cell Signaling #9860, Danvers, MA) was used according to the manufacturer's protocol. Real-time quantitative polymerase chain reaction (RT–qPCR) was carried out as we previously reported.⁶ Immunostaining and immunoblots were performed following standard protocols,⁸ using antibodies against Ki-67 (abcam #16667, Cambridge, UK), p21 (Cell Signaling, #2957), and GAPDH (Cell Signaling, #97166S). All calculations were performed using SPSS 26 (IBM, Armonk, NY), setting P < 0.05 as significant difference.

Fifty-one patients successfully completed the study with a 6-month follow-up: 26 received α-GPC (78.2 ± 7.4 years old), whereas 25 did not (77.1 ± 7.1 years old). There were no significant differences in terms of age, body mass index, blood pressure, blood glucose, serum creatinine, and therapies (Table 1). Parameters used to define frailty were distributed in a comparable manner between groups (Figure 1A). α-GPC significantly improved cognitive function at follow-up (Figure 1A). Overall, α-GPC was well tolerated and no major side effects were reported. In HUVECs, we first performed preliminary dose-response and time-course experiments (not shown); then, we observed that 10 nM α-GPC (24 h) significantly attenuated senescence induced by angiotensin II (Ang II, 1 µM), assessed measuring p21 via RT–qPCR, immunoblot, and immunofluorescence, and quantifying SA-β-gal expression (Figures 1B and 1C). Equally important, the same concentration of α-GPC improved cell proliferation, assessed by quantifying Ki-67 expression (Figure 1D), migration, measured through the wound healing test (Figure 1E, bottom), and angiogenesis, evaluated in terms of formation of network-like structures (Figure 1E, bottom).

Table 1.

Main characteristics of the study population. Data are expressed as mean ± SD or number and percentage

Parameter	Baseline			Follow-up
	Control	α−GPC	P	Control	α−GPC	P
n	26	25	—	26	25	—
BMI (kg/m2)	29.2 ± 3.3	28.9 ± 3.4	0.75	29.0 ± 3.5	28.6 ± 3.2	0.67
SBP (mmHg)	133.9 ± 11.4	132.3 ± 11.0	0.61	132.9 ± 11.1	130.8 ± 10.4	0.48
DBP (mmHg)	82.1 ± 10.1	81.4 ± 10.4	0.81	81.7 ± 10.2	80.2 ± 9.9	0.59
Heart rate (bpm)	73.7 ± 8.8	73.8 ± 9.2	0.96	73.9 ± 9.1	73.2 ± 9.3	0.78
Anti-hypertensive treatments
ACE inhibitors or ARBs, n (%)	21 (81.0)	21 (84.0)	0.78	—	—
Calcium inhibitors, n (%)	18 (69.0)	16 (64.0)	0.71	—	—
β−blockers, n (%)	19 (73.0)	18 (72.0)	0.93	—	—
Diuretics, n (%)	8 (31.0)	7 (28.0)	0.81	—	—
Laboratory analyses
Plasma glucose (mg/dL)	153.2 ± 44.0	153.4 ± 52.3	0.98	152.0 ± 46.3	151.2 ± 47.6	0.86
Creatinine (mg/dL)	1.0 ± 0.2	1.0 ± 0.2	0.99	1.0 ± 0.1	1.0 ± 0.1	0.99

ACE: Angiotensin converting enzyme; ARBs: Angiotensin Receptor Blockers; BMI: body mass index; DBP: diastolic blood pressure; SBP: systolic blood pressure.

Figure 1.

Clinical characteristics of the patients in terms of Fried criteria at baseline (A, left); α-GPC (its stick-and-ball chemical structure is shown in the inset) significantly increased the MoCA score at 6-month follow-up (A, right). 10 nM α-GPC (24 h) markedly reduced the expression of p21 triggered by 1 µM Ang II in human endothelial cells, both in terms of mRNA (left) and protein (representative immunoblots, right); floating bars indicate mean and min–max (B); 24 h incubation with 10 nM α-GPC significantly attenuated Ang II-induced cellular senescence, evaluated through p21 (top) and SA-β-gal (bottom) staining (C) and augmented endothelial cell proliferation, assessed via Ki-67 staining (D); 24 h incubation with 10 nM α-GPC significantly attenuated the impaired cell migration (E, top) and angiogenesis (E, bottom) caused by 1 µM Ang II. All values are expressed as mean ± SD unless otherwise specified; yellow scale bars: 400 μm; black scale bars: 100 μm, magenta scale bars: 50 μm; all in vitro experiments were performed at least in triplicate; *P < 0.05, **P < 0.01, ***P < 0.005, ****P < 0.001. Ang II: Angiotensin II; A.U.: Arbitrary Units; MoCA: Montreal Cognitive Assessment.

To our knowledge, this is the first study demonstrating the favorable effects of α-GPC on cognitive dysfunction in frail hypertensive older adults, also showing that α-GPC significantly attenuates Ang II-induced endothelial dysfunction. These findings are relevant especially considering the previously reported fundamental role of Ang II in the pathophysiology of cognitive impairment and vascular senescence.⁹ For instance, Anton Wellstein and collaborators recently demonstrated that low dose chronic Ang II induces senescence of endothelial cells,¹⁰ a process by which cell age and permanently stop dividing but do not die. Limitations of our study include the relatively small size of the groups in the clinical trial, albeit the n is above the value calculated a priori by power analysis, and having performed in vitro experiments only in one cell type. Further investigations, including in vivo tests, are warranted to corroborate our findings.

Author contributions

G.S. supervised the study. P.M., A.M., A.P., G.N., L.D.A., and A.D.L. performed the clinical investigation; U.K. performed the in vitro assays. U.K. and A.L. performed the statistical analysis. P.M., U.K., F.V., V.T., and G.M. drafted the manuscript. G.S. edited the manuscript. All authors were involved in the interpretation of the results and the critical review of the manuscript. All authors gave final approval and agreed to be accountable for all aspects of work ensuring integrity and accuracy.

Sources of funding

The Santulli's Lab is supported in part by the National Institutes of Health (NIH): National Heart, Lung, and Blood Institute (NHLBI: R01-HL146691, R01-HL159062, R01-HL164772, T32-HL144456), National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK: R01-DK123259, R01-DK033823), National Center for Advancing Translational Sciences (NCATS: UL1-TR002556-06, UM1-TR004400) to G.S., by the Diabetes Action Research and Education Foundation (to G.S.), and by the Monique Weill-Caulier and Irma T. Hirschl Trusts (to G.S.). U.K. is supported in part by a postdoctoral fellowship of the American Heart Association (AHA: 23POST1026190).

Data availability

The data underlying this article can be shared on reasonable request to the first author(s).