*Communication* **miR-4432 Targets FGFBP1 in Human Endothelial Cells**

**Roberta Avvisato 1,2,3,†, Pasquale Mone 1,2,†, Stanislovas S. Jankauskas 1,2 , Fahimeh Varzideh 1,2 , Urna Kansakar 1,2 , Jessica Gambardella 1,2,3, Antonio De Luca <sup>4</sup> , Alessandro Matarese <sup>5</sup> and Gaetano Santulli 1,2,3,6,7,8,\***


**Simple Summary:** The inner layer of blood vessels is formed by endothelial cells. When these cells do not work properly, several issues ensue in the human body. One of these issues is elevated blood pressure, also known as hypertension, which is an established risk factor for ischemic heart disease, stroke, chronic kidney disease, and dementia. However, the exact mechanisms linking dysfunctional endothelium and hypertension are not fully defined. In this work, we discovered that a small nucleic acid (miR-4432) is able to target and inhibit a specific gene (fibroblast growth factor binding protein 1, *FGFBP1*) in human brain microvascular endothelial cells, and we demonstrate for the first time that this miR-4432 significantly reduces endothelial oxidative stress, a well-established feature of hypertension. Taken together, our findings provide unprecedented mechanistic insights and open the field to new studies aimed at ameliorating endothelial dysfunction by harnessing miR-4432-based strategies.

**Abstract:** MicroRNAs (miRs) are small non-coding RNAs that modulate the expression of several target genes. Fibroblast growth factor binding protein 1 (FGFBP1) has been associated with endothelial dysfunction at the level of the blood–brain barrier (BBB). However, the underlying mechanisms are mostly unknown and there are no studies investigating the relationship between miRs and FGFBP1. Thus, the overarching aim of the present study was to identify and validate which miR can specifically target FGFBP1 in human brain microvascular endothelial cells, which represent the best in vitro model of the BBB. We were able to identify and validate miR-4432 as a fundamental modulator of FGFBP1 and we demonstrated that miR-4432 significantly reduces mitochondrial oxidative stress, a well-established pathophysiological hallmark of hypertension.

**Keywords:** blood–brain barrier; blood pressure; cerebrovascular disease; endothelial dysfunction; hBMECs; hypertension; HUVEC; microRNA; miRNA; miR-4432-3p

#### **1. Introduction**

Hypertension is a leading risk factor for ischemic heart disease, stroke, chronic kidney disease, and dementia [1]. It is a multifactorial disease involving interactions among genetic, environmental, demographic, vascular, and neuroendocrine factors [2,3]. Endothelial

**Citation:** Avvisato, R.; Mone, P.; Jankauskas, S.S.; Varzideh, F.; Kansakar, U.; Gambardella, J.; De Luca, A.; Matarese, A.; Santulli, G. miR-4432 Targets FGFBP1 in Human Endothelial Cells. *Biology* **2023**, *12*, 459. https://doi.org/10.3390/ biology12030459

Academic Editor: Matthias S. Leisegang

Received: 14 February 2023 Revised: 9 March 2023 Accepted: 13 March 2023 Published: 16 March 2023

**Copyright:** © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

dysfunction is an established hallmark of hypertension [4–6]; however, the exact molecular mechanisms linking dysfunctional endothelial cells (ECs) and high blood pressure are not fully understood. Several genome-wide association studies (GWAS) have identified a number of genes associated with hypertension [7,8], but only a few of these genes have been functionally validated. In 2019, the International Consortium of Antihypertensive Pharmacogenomics Studies (ICAPS) recognized fibroblast growth factor binding protein 1 (FGFBP1) as one of the genes involved in the regulation of blood pressure [9]. FGFBP1 is a key promoter of the development of the blood–brain barrier (BBB) [10], an aspect that is especially relevant considering that ECs are a major component of the BBB [11], which is crucial for maintaining neuronal and glial function [12]. Specifically, FGFBP1 has been implied in refining and maintaining barrier characteristics in the mature BBB endothelium [13]. thelial dysfunction is an established hallmark of hypertension [4–6]; however, the exact molecular mechanisms linking dysfunctional endothelial cells (ECs) and high blood pressure are not fully understood. Several genome-wide association studies (GWAS) have identified a number of genes associated with hypertension [7,8], but only a few of these genes have been functionally validated. In 2019, the International Consortium of Antihypertensive Pharmacogenomics Studies (ICAPS) recognized fibroblast growth factor binding protein 1 (FGFBP1) as one of the genes involved in the regulation of blood pressure [9]. FGFBP1 is a key promoter of the development of the blood–brain barrier (BBB) [10], an aspect that is especially relevant considering that ECs are a major component of the BBB [11], which is crucial for maintaining neuronal and glial function [12]. Specifically, FGFBP1 has been implied in refining and maintaining barrier characteristics in the mature BBB endothelium [13].

genetic, environmental, demographic, vascular, and neuroendocrine factors [2,3]. Endo-

MicroRNAs (miRs) are a relatively well conserved group of small (~21 nucleotides) non-coding RNAs that modulate the expression of their target genes: miRNAs can bind the 3 0 untranslated region (UTR) of specific genes, thereby inhibiting their expression. Thus, miRNAs have been involved in numerous pathological and physiological processes [14,15]. Others and ourselves have, in the last decades, identified a variety of miRs involved in the regulation of endothelial function [16,17]. MicroRNAs (miRs) are a relatively well conserved group of small (~21 nucleotides) non-coding RNAs that modulate the expression of their target genes: miRNAs can bind the 3′ untranslated region (UTR) of specific genes, thereby inhibiting their expression. Thus, miRNAs have been involved in numerous pathological and physiological processes [14,15]. Others and ourselves have, in the last decades, identified a variety of miRs involved in the regulation of endothelial function [16,17].

Since FGFBP1 has been previously linked to the modulation of the BBB, and precisely to endothelial function, the central scope of the present study is to detect which miR can target FGFBP1 in hBMECs (human brain microvascular endothelial cells). Since FGFBP1 has been previously linked to the modulation of the BBB, and precisely to endothelial function, the central scope of the present study is to detect which miR can target FGFBP1 in hBMECs (human brain microvascular endothelial cells).

#### **2. Results 2. Results**

#### *2.1. miR-4432 Targets FGFBP1 in a Conservative Manner 2.1. miR-4432 Targets FGFBP1 in a Conservative Manner*

*Biology* **2023**, *12*, x FOR PEER REVIEW 2 of 10

We applied bioinformatic analyses and functional experiments which led, for the first time to our knowledge, to the identification of hsa-miR-4432-3p (miR-4432) as a crucial modulator of FGFBP1 transcription, in a manner that is highly conserved across different species, including primates such as chimpanzee (*Pan troglodytes*), orangutan (*Pongo abelii*), macaque (*Macaca mulatta*), and gorilla (*Gorilla gorilla*), although it is not detected in mouse (*Mus musculus*) and rat (*Rattus norvegicus*), as shown in Figure 1. We applied bioinformatic analyses and functional experiments which led, for the first time to our knowledge, to the identification of hsa-miR-4432-3p (miR-4432) as a crucial modulator of FGFBP1 transcription, in a manner that is highly conserved across different species, including primates such as chimpanzee (*Pan troglodytes*), orangutan (*Pongo abelii*), macaque (*Macaca mulatta*), and gorilla (*Gorilla gorilla*), although it is not detected in mouse (*Mus musculus*) and rat (*Rattus norvegicus*), as shown in Figure 1.


**Figure 1.** Identification of miR-4432 as a specific modulator of FGFBP1; the complementary nucleotides between the target region of FGFBP1 30 -UTR and hsa-miR-4432-3p, highlighted in yellow, are conserved across a number of different species, including primates.

Furthermore, we designed a mutant construct of FGFBP1 30 -UTR ("FGFBP1 MUT") that harbors nucleotide substitutions at the level of the miR-4432 binding sites of FGFBP1 3 0 -UTR, as illustrated in Figure 2. Furthermore, we designed a mutant construct of FGFBP1 3′-UTR ("FGFBP1 MUT") that harbors nucleotide substitutions at the level of the miR-4432 binding sites of FGFBP1 3′-UTR, as illustrated in Figure 2. that harbors nucleotide substitutions at the level of the miR-4432 binding sites of FGFBP1 3′-UTR, as illustrated in Figure 2. that harbors nucleotide substitutions at the level of the miR-4432 binding sites of FGFBP1 3′-UTR, as illustrated in Figure 2.

Furthermore, we designed a mutant construct of FGFBP1 3′-UTR ("FGFBP1 MUT")

Furthermore, we designed a mutant construct of FGFBP1 3′-UTR ("FGFBP1 MUT")

**Figure 1.** Identification of miR-4432 as a specific modulator of FGFBP1; the complementary nucleotides between the target region of FGFBP1 3′-UTR and hsa-miR-4432-3p, highlighted in yellow, are

**Figure 1.** Identification of miR-4432 as a specific modulator of FGFBP1; the complementary nucleotides between the target region of FGFBP1 3′-UTR and hsa-miR-4432-3p, highlighted in yellow, are

**Figure 1.** Identification of miR-4432 as a specific modulator of FGFBP1; the complementary nucleotides between the target region of FGFBP1 3′-UTR and hsa-miR-4432-3p, highlighted in yellow, are
