**1. Introduction**

Hypertension is the most common chronic disease and yields considerable morbidity and mortality globally [1]. Because of the multifactorial nature of hypertension, the use of various animal models, which evoke hypertension by different mechanisms, is advantageous for unraveling disease pathogenesis and developing novel antihypertensive drugs [2–4]. Though we are seeing tremendous progress on experimental hypertension, the prevalence of hypertension is still high and continues to rise worldwide [5].

Epidemiological and animal studies support that hypertension may be programmed in utero [6–9]. The association between fetal development and increased risk of adult disease has emerged as the concept of developmental origins of health and disease (DOHaD) [10]. A wide spectrum of early-life insults can evoke developmental programming resulting hypertension later in life. These insult stimuli include, but are not limited to, maternal malnutrition (both under- and overnutrition), maternal medical conditions, environmental exposure to toxins/chemicals, lifestyle changes, and medicines taken during pregnancy [7–9,11–14].

Over the past decade, the pathogenesis behind hypertension of developmental origins has not been fully elucidated, but data from animal models have proposed several key mechanisms [14]. Until now, the proposed mechanisms consist of aberrant renin– angiotensin system (RAS), oxidative stress, reduced nephron numbers, gut microbiota

**Citation:** Tain, Y.-L.; Hsu, C.-N. Hypertension of Developmental Origins: Consideration of Gut Microbiome in Animal Models. *Biomedicines* **2022**, *10*, 875. https://doi.org/10.3390/ biomedicines10040875

Academic Editors: Josef Zicha and Ivana Vanˇeˇcková

Received: 10 March 2022 Accepted: 8 April 2022 Published: 9 April 2022

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dysbiosis, dysregulated nutrient-sensing signals, sex differences, epigenetic regulation, etc. [7–9,11–14]. Among them, the interaction between the gut microbiota and the host implicated in hypertension has received significant interest [15–18]. Despite gut microbiota dysbiosis being observed in multiple animal models of hypertension [15,16], too little attention has been paid to its role in hypertension of developmental origins.

Although blood pressure (BP) is considered with a multifactorial pattern of inheritance, genome-wide association studies cumulatively could only explain ~3.5% of BP trait variability [19]. Accordingly, it is likely that the influence of environmental and epigenetic factors on the developmental programming of hypertension should receive wider recognition. Notably, maternal insults can impair gut microbiota composition and function, leading to adverse offspring outcomes later in life [20]. Conversely, review elsewhere indicted that early-life gut microbiota-targeted therapies have benefits on the prevention of the developmental programming of adult disease, including hypertension [21]. All this raises the notion that we need to pay more attention to prevent and not just treat hypertension, with a focus on the influence of dysbiotic gut microbiota on hypertension of developmental origins. Accordingly, animal models would likely be very useful in unraveling these actions.

In this review, we describe the role of gut microbiota implicated in animal models used for studying the developmental programming of hypertension. Therefore, we summarize the contributions of animal models linking the gut microbiota to developmental programming of hypertension, which helps in developing valuable strategies to prevent hypertension from happening. We specifically focus on addressing gut microbiota-targeted therapies such as probiotics, prebiotics, and postbiotics as a reprogramming strategy for prevention of hypertension of developmental origins.

In view of the above, a search was performed in the electronic bibliographic database PubMed/MEDLINE. Search terms were as follows: "developmental programming", "DO-HaD", "animal model", "pregnancy", "gestation", "offspring", "progeny", "prenatal", "perinatal", "mother", "maternal", "reprogramming", "gut microbiota", "probiotics", "prebiotics", "postbiotics", "synbiotics", "blood pressure", and "hypertension." Relevant abstracts were identified and reviewed to identify appropriate studies. Suitable published articles in English were included, without restriction of the time of publication.

#### **2. Hypertension of Developmental Origins: Choice of Animal Models**

Compared to animal models of essential hypertension established in prior research [2,3], the etiologies of hypertension of developmental origins and underlying pathogenic mechanisms are more complex [14]. Animal models of hypertension of developmental origins can be categorized in different ways (Figure 1).

Firstly, these models can be classified according to early-life adverse conditions. Nutritional programming is the most common type of animal model being studied in the field of DOHaD research [22]. Dietary caloric or protein restriction in animals mimics the starvation linked to famine in human cohorts [23,24]. Imbalance of maternal nutrition can have longterm changes in BP, resulting hypertension in adult offspring [25]. Inadequate or excessive intake of a specific nutrient has been established to induce hypertension of developmental origins in animal models, as reviewed elsewhere [11,26]. These models of undernutrition related to hypertension of developmental origins include, but are not limited to, caloric restriction [27], protein restriction [28], and deficiencies in sodium [29], calcium [30], zinc [31], iron [32], methyl donor nutrients (choline; vitamins B2, B6, and B12; folic acid; and methionine) [33], and vitamin D [34]. On the other hand, overnutrition characterized by the consumption of a high-fat [35,36], high-fructose [37,38], or high-protein diet [39] by rodent mothers also leads to early programming of hypertension in the offspring. Additionally, animal models resembling maternal medical conditions have also been evaluated in developmental programming of hypertension. These models include hypertensive disorders of pregnancy [40], preeclampsia [41], diabetes [42], chronic kidney disease (CKD) [43], maternal hypoxia [44], etc. Furthermore, chemical and medication exposures during pregnancy increase the risk of developing hypertension in offspring [13,14]. Prenatal exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) [45], bisphenol A [46], nicotine [47], caffeine [48], cyclosporine [49], gentamicin [50], tenofovir [51], minocycline [52], or glucocorticoids [53] has been reported to induce hypertension of developmental origins in various animal models.

**Figure 1.** The classification of major animal models for studying hypertension of developmental origins.

Secondly, animal models can be classified based on molecular mechanisms. In view of different early-life adverse environmental factors producing the same outcome, that is to say hypertension in adult offspring, there might be core mechanisms underlying hypertension of developmental origins. These mechanisms include gut microbiota dysbiosis [21], oxidative stress [12], aberrant RAS [54], reduced nephron numbers [7], dysregulated nutrient-sensing signals [55], sex differences [56,57], epigenetic regulation [58], inflammation [9,14], nitric oxide (NO) deficiency [59], etc. Up to date, various animal models have been developed to test such proposed mechanisms. Because of the multifactorial nature of developmental hypertension, the use of various animal models, each of which induces hypertension by a different mechanism yet with the same end result, is advantageous. This approach would allow for a novel and effective reprogramming intervention targeting a specific molecular pathway to be adopted for preventions and therapies.

Lastly, animal models in DOHaD research can be classified according to species [60]. Diverse large- and small-animal models have been used, each with its own natural advantages and disadvantages [8]. Former reviews demonstrated that cow [61], sheep [62], rat [27], and mice [63] have be used to study hypertension of developmental origins [14]. Considering that rat models are cost-effective and easy to maintain and breed, they became the most common species used in the research field of DOHaD-related hypertension [14]. Although nonhuman primates [64], swine [65], rabbits [66], and guinea pig [67] have been studied for cardiovascular outcomes induced by maternal insult stimuli, none of them have been reported for examining hypertension of developmental origins.

Rats are by far the most often used species in the field of primary hypertension research. Of these, the spontaneously hypertensive rat (SHR) without any doubt is the most popular strain [2]. However, the majority of the rat strains used for studying developmental hypertension are Sprague-Dawley (SD) or Wistar [14]. In view of the genetic background of SHR, offspring develops hypertension spontaneously without programming induced by early-life insults, weakening its application in studying hypertension of developmental origins. Hence, the choices of animal models between essential hypertension and hypertension of developmental origins are quite different. Many more aspects of animal models need to be taken into further consideration, such as the timing of organogenesis [68], life cycle [69], gestation period [70], litter size [71], offspring outcomes, than in human studies [72], and valuable therapeutic interventions need to be evaluated and validated [14].

Together, it is noted that remarkable advances in hypertension of developmental origins have been originated from animal models. However, what is missing in the literature is animal models used for studying hypertension-related complications. Although elevated BP is the core feature of human hypertension, its morbidity and mortality occur with complications in the heart, brain, kidneys, and vessels. The contributions of early-life insults to these hypertension-related complications later in life in an organ-dependent manner have not yet been well-studied in the above-mentioned animal models.

#### **3. Gut Microbiota: Choice of Animal Models**

Trillions of bacteria living in the gut—the gut microbiota—coexist with the host in a mutually beneficial relationship [73]. Microbiota refers to all the microorganisms found in the environment, while the term microbiome refers to the collection of genomes from all microorganisms in a given environment. A variety of environmental factors can cause the disturbance of gut microbiota (i.e., dysbiosis), which in turn can influence human health and disease. Although the influence of gut microbiota in hypertension has been extensively reviewed elsewhere [15–18], less attention was paid to exploring its role in hypertension of developmental origins.

Directly after birth, microbes colonize the neonatal gut immediately [74]. These alterations continue until three years of age and mediate the transition toward an adult-like gut microbiota [75]. During pregnancy and lactation, the mothers share gut microbes and microbial metabolites with their offspring, which highlights the importance of maternal influences in the development of early-life gut microbiota [76]. A diversity of early-life factors governs the establishment of the gut microbiota, such as maternal medical conditions, gestational age, types of delivery, antibiotic exposure, formula feeding, and ecological factors [74–77].

So far, animal models have been broadly established to investigate human diseases in gut microbiota research [78]. Figure 2 illustrates various approaches to alter the gut microbiota in animal models of disease. Several gut microbiota-targeted therapies have been used to alter gut microbiota compositions and its derived metabolites. These interventions consist of probiotics, prebiotics, synbiotics, postbiotics, etc. [14]. The embryo transfer (ET) method is considered the gold standard for gut microbiota transfer. Additionally, researchers often use other methods to transfer the gut microbiota, such as fecal microbiota transfer (FMT), co-housing (CH), or cross-fostering (CF) [78].

Several gut microbiota-targeted therapies have shown to alter the gut microbiome. Probiotics (i.e., live beneficial microbes) and prebiotics (i.e., substances in foods that promote the growth of healthy microbes) are the most commonly used gut microbiota-targeted modalities in clinical practice [79]. Synbiotics refer to a mixture comprising probiotics and prebiotics that also confers a health benefit [79]. In addition, the use of substances leased or produced through gut microbial metabolism, namely postbiotics, have shown an influence on gut microbiota compositions and metabolites [80].

**Figure 2.** Different approaches to altering the gut microbiota. (**A**) Gut microbiota-target therapy; (**B**) embryo transfer; (**C**) fecal microbiota transfer; (**D**) cross-foster; (**E**) co-house. FMT = fecal microbiota transfer; GM = gut microbiota; GM1 = transferred gut microbiota; GM2 = GM + GM1.

In the approach to transfer embryos, they are collected from the gut microbiome recipient and surgically transferred to a pseudopregnant donor dam [78]. Accordingly, the recipient pups obtain the vaginal microbiota from the donor dam through vaginal delivery. Nevertheless, this method needs considerable costs and expertise, making it inaccessible for many labs. Using the FMT approach, feces or fecal contents from donors are transferred to recipient animals via gastric gavage. Germ-free mice or antibiotics-treated depleted microbiota animals are commonly used as recipients [81].

Another commonly used method is CH, wherein recipients are co-housed with a donor after weaning [82], leading to the transfer of the donor gut microbiome through coprophagy and grooming [82]. Although the co-housing approach is easy and low-cost, the transfer of the gut microbiota after the critical developmental period results an incomplete transfer as well as a hybridized gut microbiome. When the recipient pups are housed in cages with the donor dam within 24 h after birth, the CF method allows the recipients to obtain most of their gut microbiota from the donor dam [78]. Compared to CH, the CF approach transfers the gut microbiota from an early age during the maternal care process.

All these methods each carry certain advantages and limitations. Researchers should thus be mindful of these method-related differences in the context of the transfer methods used for studying the role of the gut microbiota on hypertension of developmental origins.

#### **4. Gut Microbiota in Hypertension of Developmental Origins**

There is mounting evidence to support the pathogenic interconnection between the gut microbiome and hypertension [15–18]. However, there is paucity of information regarding the influence of the gut microbiota on the developmental programming of hypertension later in life. Therefore, most data obtained from patients with established hypertension and knowledge received from animal models of essential hypertension might be extrapolated to hypertension of developmental origins.

#### *4.1. Gut Microbiota and BP Regulation*

A great deal of work on the influence of the gut microbiota and its derived metabolites on BP regulation has been conducted. First, data from several genetic hypertensive rat models (e.g., SHR) indicated that the gut microbiota of hypertensive rats is dysbiotic and significantly different from the microbiota of normotensive control rats [15]. Gut microbiota dysbiosis was also noted for other hypertension models such as animals treated with high salt [83], angiotensin II [84], and deoxycorticosterone acetate-salt [85]. Another line of evidence comes from germ-free animals. The absence of microbiota in germ-free rats resulted with relative hypotension compared with their conventionalized counterparts, suggesting an essential role of gut microbiota in BP regulation [86]. Additionally, germ-free mice that received FMT from a hypertensive human donor developed a gut microbiota similar to that of their donor, as well as elevated BP [87]. There are observations that microbial metabolites are involved in BP homeostasis. Short chain fatty acids (SCFAs) are the main metabolites produced during bacterial fermentation of carbohydrates. SCFAs are generally known to regulate BP via activating their SCFA receptor, including olfactory receptor 78 (Olfr78), G protein-coupled receptors (GPR) GPR41, GPR43, and GRP109A [88]. Another example is trimethylamine-N-oxide (TMAO). TMAO is a small colorless amine oxide produced by gut microbiota metabolism [89]. A high TMAO level correlates with CVD mortality [90]. Fourth, the uses of probiotics [91] or prebiotics [92] have shown benefits on hypertensive patients.

#### *4.2. Animal Models Linking Gut Microbiota Dysbiosis to Hypertension of Developmental Origins*

Much work investigating the actions of the gut microbiome has directly studied the hypertension models, yet relatively little data exists on its programming effect related to hypertension of developmental origins. A summary of animal studies indicating the association between dysbiotic gut microbiota and developmental hypertension in adult offspring is provided in Table 1 [40,52,93–106].

The current review is only restricted to early-life insults starting in the pregnancy and/or lactation period. Table 1 shows that rats are the most common species being used. A variety of early-life insults have been reported to induce developmental hypertension, accompanying alterations of the gut microbiota, including a maternal high-fructose diet [93–95], maternal high-fructose diet plus TCDD exposure [96], maternal high-fat/highcholesterol diet [97], maternal high-fat and/or post-weaning high-fat diet [98,99], gestational hypertension [40,100], maternal CKD [43], maternal dyslipidemia [101], maternal NG-nitro-L-arginine-methyl ester (L-NAME) administration plus postnatal high-fat diet [102], maternal administration of minocycline [52], maternal TMAO and asymmetric dimethylarginine (ADMA) exposure [103], maternal TCDD exposure [104,105], and prenatal androgen exposure [106].

Table 1 lists the timing of hypertension determined from rat models, with age ranging from 12 weeks to four months. As every month of an adult rat corresponds to three human years [55], the observed periods correspond with humans from childhood to early adulthood.

#### *4.3. Gut Microbiota Dysbiosis in Hypertension of Developmental Origins*

The study of the gut microbiome in animal models of developmental hypertension mainly focuses on four types of dysbiosis: loss of diversity, decreases in beneficial microbes, shifts in key taxa, and alterations of microbial metabolites. A schematic summarizing the gut microbiota and a possible molecular pathway linked to hypertension of developmental origins is presented in Figure 3.


**Table 1.** Animal models reporting hypertension of developmental origins associated with dysbiotic gut microbiota.

Studies tabulated according to animal models, and age at measure; SD = Sprague-Dawley; SHR = spontaneously hypertensive rat; M = male; F = female; CKD = chronic kidney disease; TCDD = 2,3,7,8-tetrachlorodibenzo-pdioxin; ADMA = asymmetric dimethylarginine; GPR41 = G protein-coupled receptor 41; GPR43 = G proteincoupled receptor 43; TNA = trimethylamine; TMAO = trimethylamine N-oxide; L-NAME = NG-nitro-L-argininemethyl ester; F/B ratio = *Firmicutes* to *Bacteroidetes* (F/B) ratio; SCFA = short chain fatty acid.

**Figure 3.** Overview of the gut microbiota and potential molecular mechanisms related to hypertension of developmental origins. SCFA short chain fatty acid. TMAO = trimethylamine N-oxide; TMA = trimethylamine; SCFA = short chain fatty acid; RAS = renin-angiotensin system; TH17 = T helper 17 cells; TH1 = T helper 1 cells; F/B ratio = *Firmicutes* to *Bacteroidetes* ratio; AhR = aryl hydrocarbon receptor.

## 4.3.1. Alterations in Gut Microbiota Compositions

First, α-diversity is decreased in models of maternal high-fat and high-cholesterol diet [97] and maternal TCDD exposure [104,105]. A similar pattern of gut dysbiosis was reported in several hypertensive animal models [15]. Second, a maternal plus post-weaning high-fat diet programming offspring's hypertension coincides with an increased *Firmicutes* to *Bacteroidetes* (F/B) ratio and a reduction of genera *Lactobacillus* and *Akkermansia* [98,99]. This was found to be consistent with hypertension models showing the F/B ratio was increased and served as a microbial marker of hypertension [15]. Likewise, the increase of the F/B ratio is noted in other models of developmental hypertension programmed by a variety of maternal insults, including CKD [43], minocycline administration [52], hypertension [100], L-NAME administration plus high-fat diet [102], and TCDD exposure [104,105]. Both *Akkermansia* and *Lactobacillus* are known as one of the beneficial probiotic bacterial strains [107,108]. Decreases of certain beneficial microbes were also found in developmental models of hypertension, like maternal minocycline administration [52], maternal high-fructose diet [94], maternal hypertension [40], maternal dyslipidemia [101], and maternal TCDD exposure [104,105].
