4.4.2. Aberrant RAS

The RAS is a major regulatory network that maintains BP, and blockade of the RAS has emerged as a therapeutic option for hypertension [122]. An increasing number of animal models linked to aberrant RAS are now being developed to evaluate hypertension of developmental programming, as reviewed elsewhere [54].

Within the RAS, regulation is achieved through a cascade of proteases generating some bioactive peptides [122]. The classical RAS consists of angiotensin-converting enzyme (ACE), angiotensin (ANG) II, and angiotensin II type 1 receptor (AT1R). Activation of the classical RAS elicits vasoconstriction and inflammation under pathophysiological conditions, consequently resulting in hypertension and its related complications [123].

On the other hand, the nonclassical RAS is composed of ACE2/angiotensin-(1-7) (Ang-(1-7))/Mas receptor/ANG II type 2 receptor (AT2R), by which it can counterbalance the adverse effects of ANG II [124]. A growing body of evidence supports that aberrant RAS plays a key role in developmental hypertension, and RAS-based interventions can be used as a reprogramming strategy to prevent hypertension [54].

Mounting evidence suggests a bidirectional interaction between the gut microbiota and RAS; alterations in RAS shift microbiota composition and metabolic activity, while gut microbiota-derived metabolites can modulate the gut RAS [125]. Through regulation of intestinal amino acid transport, prior research reported that ACE2 plays a key non-catalytic role in gut biology and modulation of the gut microbiota [126].

Adult rat progeny of CKD mothers developed hypertension, coinciding with decreased expression of Mas receptor and AT2R [43]. In another high-fructose diet plus TCDD exposure model, 3,3-dimethyl-1-butanol (DMB) therapy protected against hypertension, coinciding with a reduction of AT1R but an increase of AT2R protein abundance, as well as reshaping the gut microbiota [96]. Other developmental hypertension models such as a perinatal high-fat diet [99], maternal administration of minocycline [52], and maternal TMAO plus ADMA exposure [103] also interfere with aberrant RAS and gut microbiota dysbiosis.

As gut microbiota dysbiosis has been linked to hypertension by modulating the systemic and local RAS [127], these observations endorse the idea that the interaction between the RAS and the gut microbiota implicates the pathogenesis of developmental programming of hypertension, although this remains speculative.

#### 4.4.3. Inflammation and Immune Response

Pregnancy is considered a physiologic systemic inflammatory response; compromised pregnancies and associated complications may be attributed to inflammation [128]. The accumulation of T cells, monocyte/macrophages, and T cell-derived cytokines is involved in the pathogenesis of hypertension [129]. An imbalance of T regulatory cells (Treg) and T helper 17 (TH17) cells has been linked to hypertension [129], which can be restored by postbiotic therapy [130]. In CKD, the interplay between Treg/TH17 balance and inflammation has also been related to hypertension [131]. Treg and TH17 cells can both be regulated by aryl hydrocarbon receptor (AhR) [132].

It is noted that several microbial tryptophan metabolites are uremic toxins as well as AhR ligands. AhR signaling can initiate inflammation through increasing monocyte adhesion, upregulating proinflammatory gene expression, reducing NO bioavailability, and inducing the expression of endothelial adhesion molecules [133]. Several gut microbiotaderived uremic toxins, like indoleacetic acid and indoxyl sulfate, have pro-oxidant, proinflammatory, procoagulant, and proapoptotic effects, all of which are involved in the pathogenesis of hypertension [134].

Using a rat model of maternal CKD-induced hypertension, we observed that maternal tryptophan therapy preventing offspring's hypertension coincides with restoration of the AhR signaling pathway and several tryptophan-metabolizing microbes [134]. Another study showed that TCDD-induced programming hypertension is related to TH17-induced renal inflammation, the activation of AhR signaling, and alterations of gut microbiota compositions [105]. In contrast, TCDD-induced activation of AhR signaling and TH17 responses can be restored by perinatal supplementation with resveratrol, an AhR antagonist. Additionally, resveratrol is reported to have benefits on offspring hypertension in several developmental hypertension models [45,46,95,102].

Although results from animal models support the role of inflammation and immunity on hypertension of developmental origins, more research is required to gain comprehensive insight into their interconnections with the gut microbiota and develop therapeutic potential of inflammation- or immune-targeted therapies in hypertension of developmental origins and associated organ damage.

#### **5. Reprogramming Strategy: Gut Microbiota-Targeted Therapy**

The idea from DOHaD research creates opportunities to reverse the programming process, namely reprogramming, by early intervention aiming to prevent hypertension of developmental origins later in life [135]. Current literature on animal studies for hypertension of developmental origins supports that gut microbiota-targeted therapy can work as a reprogramming strategy to prevent hypertension induced by various early-life insult stimuli.

#### *Animal Models Used for Reprogramming*

Here, we show Table 2 that summarizes studies documenting microbiota-targeted reprogramming therapies in animal models of developmental hypertension, restricting those starting before or upon disease onset [93,94,96,99,103,105,110,113,134,136,137]. The therapeutic duration is from pregnancy through lactation, which cover the periods of organogenesis. The literature review states that gut microbiota-targeted strategies used to prevent hypertension include probiotics, prebiotics, postbiotics, and dietary nutrients.

Table 2 illustrates that the most commonly used species are rats. Several models of developmental hypertension have been used to examine gut microbiota-targeted interventions, such as maternal high-fructose diet [93,94], perinatal high-fat diet [99,110,136], perinatal TCDD exposure [105], maternal adenine-induced CKD [113,134,137], maternal TMAO and ADMA exposure [103], and maternal high-fructose intake plus TCDD exposure [96].

Despite probiotics and prebiotics showing benefits in hypertension [91,92], there was very limited evidence in regard to their role on hypertension of developmental origins. Probiotic treatment with *Lactobacillus casei* in pregnancy and lactation prevents the development of hypertension in adult male rat offspring raised on a maternal high-fructose diet [93] or perinatal high-fat diet model [99].

As a prebiotic, inulin has a protective effect in hypertension of developmental origins [93,99]. A previous study using a maternal high-fructose model demonstrated that perinatal inulin treatment protects against offspring's hypertension, accompanied by increased abundance of *Lactobacillus*, the most-known probiotic strain [93]. Another study demonstrated that perinatal supplementing with inulin protects against maternal highfructose-diet-induced hypertension, accompanied by increases of plasma propionate concentrations [99].


**Table 2.** Summary of animal models documenting gut microbiota-targeted therapies for hypertension of developmental origins.

Studies tabulated based on types of intervention and animal models. TCDD = 2,3,7,8-tetrachlorodibenzo-p-dioxin; CKD = chronic kidney disease; TMAO = trimethylamine-N-oxide; ADMA = asymmetric dimethylarginine; SD = Sprague-Dawley rat; DMB = 3,3-maternal dimethyl-1-butanol.

Additionally, resveratrol could be used to protect against adult disease of developmental origins due to its probiotic properties [138]. Studies using a maternal TMAO plus ADMA exposure rat model indicate that adult rat progeny born to dams exposed to uremic toxins develop hypertension [103]. Nonetheless, maternal resveratrol therapy rescues from hypertension programmed by TMAO plus ADMA exposure, accompanied by increased butyrate-producing bacteria and fecal butyrate level.

Another study demonstrated that adult rat progeny born to dams exposed to TCDD have hypertension [105], which is related to activation of AhR signaling, induction of TH17 dependent renal inflammation, and shifts of gut microbiota compositions [105]. Conversely, the induction of TH17- and AhR-mediated inflammation can be counterbalanced by perinatal resveratrol supplementation. The beneficial effects of resveratrol are also relevant to reshaping the gut microbiota by augmenting microbes that can inhibit TH17 responses and reducing the F/B ratio. In a maternal CKD model, adult rat progeny developed renal hypertrophy and hypertension [82]. Perinatal resveratrol therapy protects from hypertension, accompanied by restoration of microbial richness and diversity and an increase in beneficial microbes, *Lactobacillus* and *Bifidobacterium* [113]. Nevertheless, the low bioavailability of resveratrol limits its clinical translation [139]. In this regard, we synthesized resveratrol butyrate ester

(RBE) from resveratrol and butyrate by esterification to improve the efficacy [140]. We recently found that low-dose RBE (25 mg/L) is as effective as resveratrol (50 mg/L) in protecting against CKD-induced hypertension [141], despite the beneficial effect of RBE in models of developmental hypertension awaiting further evaluation.

Although there are many other prebiotic foods, only garlic oil has shown benefits on protection of perinatal high-fat-diet-induced hypertension in adult progeny [110]. The beneficial effects of garlic oil include increased α-diversity; increased plasma acetate, butyrate, and propionate; and increased beneficial bacteria *Lactobacillus* and *Bifidobacterium*.

In addition to probiotics and prebiotics, postbiotics are another gut microbiota-targeted modality. Postbiotics include various components like microbial cell fractions, extracellular vesicles, cell lysates, extracellular polysaccharides, functional proteins, cell wall-derived muropeptides, etc. [80]. Nevertheless, very limited information exists regarding the use of postbiotics in hypertension. As a postbiotic, acetate for perinatal supplementing showed benefits on maternal high-fructose-diet-induced hypertension [94].

Additionally, two studies demonstrated that maternal DMB therapy, an inhibitor of TMA formation, protects from hypertension in adult rat progeny exposed to maternal highfructose diet with or without TCDD exposure [94,96]. Its beneficial effect was accompanied by affection of the metabolic pathway of TMA-TMAO and reshaping the gut microbiota. Another example of postbiotics use for hypertension of developmental origins is conjugated linoleic acid. One study showed the benefit of conjugated linoleic acid, a gut microbial metabolite derived from dietary polyunsaturated fatty acids, on high-fat-diet-induced hypertension [136].

Moreover, dietary nutrients have also been applied as gut microbiota-targeted therapies for hypertension of developmental origins. Former reviews have sufficiently illustrated the impact of diet on the gut microbiome [142,143]. Prior research demonstrating that specific nutrient intake can be beneficial to protecting from hypertension of developmental origins in various animal models [26,144]. These nutrients include folic acid [145], vitamin E [146], polyunsaturated fatty acids [147], and certain amino acids [27,148,149]. However, very few of them have been studied about their impact in gut microbiota related to developmental hypertension. One recent study showed that L-cysteine therapy protects adult offspring against maternal CKD-induced hypertension, associated with enhancement of beneficial genera *Oscillibacter* and *Butyricicoccus*, as well as depletion of indole-producing genera *Alistipes* and *Akkermansia* [137]. Another study reported that tryptophan supplementation during gestation prevents maternal CKD-induced offspring hypertension. The protective effect of tryptophan supplementation is related to alterations to several tryptophan-metabolizing microbes and the AHR signaling pathway [134].

Notably, increasing evidence supports the notion that altered gut microbial composition and function is evident in hypertension that can be evoked with diets high in salt [83,150–152]. Although maternal high salt consumption has been associated with offspring hypertension [29], much remains to be elucidated about the interplay between gut microbiota with high-sodium diets and the role of low-salt diet as a reprogramming strategy for hypertension of developmental origins.

Together, current evidence from animal models supports that modulation of gut microbiota compositions and its derived metabolites through gut microbiota-targeted therapies, in the long term, may enable the capacity to prevent the development of hypertension in a desired favorable direction.

## **6. Conclusions and Perspectives**

Animal models have made significant contributions to research in hypertension of developmental origins, giving rise to substantial evidence of an interconnection between early-life insult stimuli, gut microbiota dysbiosis, and hypertension in adulthood. Our review provides insights into the importance of animal models not only in investigating underlying mechanisms behind hypertension of developmental origins, but also in developing early-life gut microbiota-targeted therapy to help prevent hypertension in later life. Considering that hypertension is a major hallmark of metabolic syndrome, aforementioned animal models may be potential models to evaluate developmental programming of metabolic syndrome-related disorders, not to mention that the link between gut microbiota and metabolic syndrome have been extensively discussed in many research papers [153,154].

Animal models are generally considered an intermediate step between bench and human trials. While animal studies are a regulatory requirement for validating preliminary experimental data, animals will remain indispensable in research for some time [155]. However, alternative approaches to animal models need to be investigated and adopted. The integration of computer models with modeling in vitro tissues and organs should be considered as alternative protocols to reduce the use of animals in scientific research [156].

To move the field forward, some unsolved aspects toward clinical translation need to be considered. Despite abovementioned early-life insults having been identified in animal models of developmental hypertension, there may be more risk factors in nature that can adversely influence the BP of adult progeny awaiting to be discovered.

Another important aspect is that the implication of the gut microbiota transfer early in life to the development of hypertension is still unknown, although FMT has been extensively studied in microbiome-associated pathologies, including hypertension [78,81,82]. However, currently, little information exists about their potential application in hypertension of developmental origins.

Animal studies suggest that early use of certain prebiotics, probiotics, or postbiotics may prevent the developmental programming of hypertension, while the exact mechanisms have not been entirely elucidated. What is absent in the literature is whether other prebiotic-rich foods or prebiotic-like components, either individually or in combination, in pregnancy and lactation can also change the gut microbiota to protect adult progeny against hypertension in various animal models.

In conclusion, gut microbiota dysbiosis is a meaningfully pathogenetic link for hypertension of developmental origins. Each of the aforementioned animal models was applied to examine a specific hypothesis, and neither can be considered superior in respect of all aspects of research on hypertension of developmental origins. After all this greater understanding of animal models used for DOHaD research and remarkable growth in gut microbiota-targeted therapies, we believe that translating this growing body of evidence into clinical practice is a valuable strategy to reduce the global hypertension-related burden.

**Author Contributions:** Conceptualization, C.-N.H. and Y.-L.T.; data curation, C.-N.H. and Y.-L.T.; funding acquisition, Y.-L.T.; project administration, C.-N.H. and Y.-L.T.; writing-original draft, C.-N.H. and Y.-L.T.; writing-review and editing, C.-N.H. and Y.-L.T. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was supported by the Chang Gung Memorial Hospital, Kaohsiung, Taiwan, grants CORPG8M0201, CORPG8M0151, and CORPG8M0081, and the Ministry of Science and Technology, Taiwan, grants MOST 110-2314-B-182-020-MY3 (Y.-L.T.) and MOST 110-2314-B-182A-029 (C.-N.H.).

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Data are contained within the article.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**

