Abstract
The commensal microbiota resides in a mutualistic relationship within the mammalian gut. It significantly influences the formation of capillary networks in the small intestine, not only during development but also in adulthood. Mucosal capillaries in small intestinal villus structures play a critical role for the uptake of dietary nutrients and immune regulation. Emerging studies have elucidated how the composition of gut microbiota can influence not only postnatal gut development regarding immune tolerance, nutrient absorption, and morphology but also the development and maintenance of blood and lymphatic capillaries within the small intestine. In particular, the analysis of gnotobiotic mouse models affirmed the importance of the gut microbiota, or even only single gut bacteria, in the remodeling of the small intestinal capillaries. Here, different epithelial-to-endothelial cross talk pathways, e.g. Paneth cell-derived signals, Toll-like receptor signaling, or tissue factor–protease activated receptor-1 signaling, have been reported to affect intestinal villus vascular remodeling in a microbiota-dependent fashion. In this review article, we will provide a comprehensive overview on the relevant microbiota–host interaction pathways, which have been revealed to influence angiogenesis and vascular remodeling in the small intestine.
Introduction
The small intestinal microbiota
The adult human gut accommodates trillions of microbes, predominantly including bacteria but also fungi, viruses, and archaea, which account for the gut microbiota (Bäckhed et al. 2005a, Turnbaugh et al. 2007, Neish 2009, Thursby & Juge 2017). The entity of microbial genes present in this ecosystem is known as the microbiome (Lederberg & McCray 2001, Hooper et al. 2002, Qin et al. 2010). Residing in the gut lumen, the microbiota is in close contact to host cells. Thus, a tight regulation and maintenance of homeostasis of the intestinal mucosa is crucial. This microbial ecosystem has evolved into a mutualistic relationship with its host, forming a complex metaorganism (Hooper et al. 2002, Bäckhed et al. 2005a, Esser et al. 2019). The composition of the intestinal microbiota was reported to vary remarkably between individuals (Eckburg et al. 2005, Adak & Khan 2019). Hence, to date, it is not possible to give absolute numbers or ratios of a healthy bacterial composition (Rinninella et al. 2019). It was observed under healthy conditions that the gut microbiota is dominated by the bacterial phyla of Firmicutes and Bacteroidetes, comprising 90% of the gut commensal bacteria, while also Actinobacteria, Proteobacteria, Fusobacteria, Cyanobacteria, and Verrucomicrobia represent parts of the gut microbiome (Bäckhed et al. 2005a, Arumugam et al. 2011, Adak & Khan 2019). As part of the mutual symbiosis between the gut microbiota and their host, it performs essential functions for the host including degradation of dietary components, facilitating the availability of nutrients and crucial regulation of a myriad of host metabolic and immune pathways, as well as morphological changes like the villus vascular remodeling, while receiving an adapted niche in this holobiont system (Hooper et al. 2002, Stappenbeck et al. 2002, Bäckhed et al. 2005a, Schroeder & Bäckhed 2016, Koh & Bäckhed 2020). However, the complex and crucial interactions between the host and the gut-resident microbiota are limitedly understood.
Means of analyzing microbiota–host interactions
To study microbiota–host interactions, the use of gnotobiotic model organisms, especially the axenic (germ-free, GF) model, is irreplaceable. The gnotobiotic model is defined as an organism that is associated with none or only specific strains of microorganisms (Dremova et al. 2023). To achieve a complete GF state, these animals are generated via a rederivation process involving a sterile embryo transfer followed by foster rearing in a completely sterile isolator. When comparing GF mice with conventionally raised (CONV-R) mice, it becomes clear to what extent commensal bacteria affect host physiology and morphology (Bayer et al. 2021). GF mice exhibit a multitude of altered anatomical and physiological features, compared to CONV-R mice, including nutrition, metabolism, circulation, and intestinal functions (Kostic et al. 2013, Al-Asmakh & Zadjali 2015). Furthermore, the association of GF mice with a CONV-R microbiota, resulting in so-called conventional-derived (CONV-D) mice, allows for the observation of spontaneous changes occurring in the gut of these mice caused by the colonizing microbiota. The gnotobiotic model is superior compared to the partial ablation of microbiota by antibiotic treatment because gnotobiotics enable the analysis of mice that were never exposed to any kind of microorganism or individually colonizing GF mice with certain bacterial strains by performing monocolonization experiments (Bayer et al. 2019).
Gut physiology is influenced by the commensal microbiota
Intrinsic and extrinsic factors have been described to influence several aspects of intestinal morphology and physiology. Intrinsic factors include genetics, aging, and host cellular signaling, while extrinsic factors encompass diet, microbiota, infections, and environmental exposures. For example, high-fat diet is an extrinsic nutritional factor that was demonstrated to increase mucosal thickness and villus length in the small intestine (Todorov et al. 2020). In contrast, epithelial-to-mesenchymal signaling by endogenous intrinsically synthetized morphogens, such as hedgehog (Hh) morphogens, defines many aspects of intestinal function (van den Brink 2007). During embryonic development, the intrinsic genetic program is the main regulator of gut morphogenesis. For instance, the Hh signaling pathway has been shown to be indispensable for later stages of gastrulation and the left–right axis formation (Zhang et al. 2001, van den Brink 2007). Mutant mice lacking both Indian Hh (Ihh) and Sonic Hh (Shh) display a gut malrotation (Ramalho-Santos et al. 2000), while Shh was shown to be crucially involved in the differentiation and patterning of the intestine (Apelqvist et al. 1997). Intact Hh signaling from the gut epithelium to the mesenchyme is needed at the embryonic stage to form villus structures (Walton et al. 2012). In the adult mouse intestine, the gut microbiota suppresses Hh signaling via innate immune pathways, thus weakening the gut epithelial barrier (Pontarollo et al. 2023). Embryonic gut development has been described in detail elsewhere (van den Brink 2007, Bayer et al. 2021).
In fact, intrinsic and extrinsic factors act in concert regulating small intestinal development. Thus, it is not always possible to discriminate the initial trigger originating from the host’s intrinsic program or from the environment. For example, the gut microbiota is known to influence gene expression in host cells while in turn being subject to host cell signaling and regulation. Of note, the microbiota reshapes the small intestine not only during infancy but also during adulthood, resulting in the continual remodeling of gut morphology. Thus, the dynamic relationship between host and its resident microbes highlights the profound influence that gut microbiota wields over various aspects of our health. The modulation of the small intestinal vasculature is one of the fundamental influences the microbiota has on its host, involving distinct epithelial-to-endothelial cross talk. This review provides insight into regulatory pathways and signaling molecules that are involved in microbiota–host interactions and are relevant to initiate the remodeling of the small intestinal microcirculation.
The small intestine and its microbial residents
Functional anatomy of the small intestine
The human gut is an essential part of the gastrointestinal tract. It is divided into the esophagus, stomach, small intestine, cecum, colon, rectum, and anus, each of which fulfills its own specialized functions (Fig. 1A). Among the vital functions of the gastrointestinal tract are digestion, nutrient uptake and transport, gut barrier function, excretion and detoxification of catabolites, and protection from pathogens (Khandagale & Reinhardt 2018). The bulk of the nutrient absorption from the digested food passing through the gut lumen occurs in the small intestine. Hence, its inner surface is lined with fingerlike projections, the small intestinal villus structures, which greatly increase the surface area for nutrient absorption (Helander & Fändriks 2014). Each villus is covered by a layer of intestinal epithelial cells (IECs), comprising specialized subtypes with distinct functions (Fig. 1B). Enterocytes, which are primarily responsible for nutrient absorption, are characterized by a microvillus brush border on their apical surface enhancing their capacity for nutrient absorption of carbohydrates, proteins, fats, vitamins, minerals, and water (Kiela & Ghishan 2016). Enterocytes comprise an essential player in intestinal immune tolerance regulation (Miron & Cristea 2012). The primary function of goblet cells is the production and secretion of mucus, which comprises an important protective layer covering the intestinal epithelium to shield the host cells from invading microbes, while still allowing the active interaction between IECs and the gut commensals (Hansson 2020). The enteroendocrine cells are specialized on the production and secretion of hormones, thereby regulating various digestive and metabolic processes. The Lieberkühn crypts are found at the base of each villus harboring Paneth cells, which secrete antimicrobial peptides and lysozyme. Pluripotent stem cells are located to the crypt base, giving rise to new epithelial cells (Cheng & Leblond 1974, Umar 2010) to replace damaged intestinal epithelial cells (van der Flier & Clevers 2009, Crawley et al. 2014). The newly generated epithelial cells shift toward the villus tip, where they detach into the gut lumen, resulting in the complete renewal of the small intestinal epithelium every 3–5 days (Hall et al. 1994, van der Flier & Clevers 2009, Park et al. 2016). The lamina propria is located underneath the barrier of the small intestinal epithelium and consists of fibroblasts, myofibroblasts, smooth muscle cells (SMCs), immune cells, neurons, and endothelial cells (Fig. 1B).
Schematic view of the small intestinal villus structure showing crypts (harboring stem cells and Paneth cells) and finger-like projections covered by different IEC types, including enterocytes, goblet cells, and enteroendocrine cells. Underneath the epithelium lies the lamina propria with the vascularization network lined by endothelial cells and projecting into the villi. Each villus contains anastomosing blood capillaries enclosing a central lacteal for lymphatic supply. The commensal microbiota resides in the gut lumen and interact with host IECs. Created with BioRender.com.
Citation: Microbiota and Host 2, 1; 10.1530/MAH-23-0021
The integrity of the gut epithelial lining is crucial to maintain barrier function in homeostatic conditions, preventing invading pathogens and their constituents to translocate into the underlying tissue. In case of a disrupted barrier, microorganisms can penetrate the impaired gut epithelial boarder and trigger inflammation resulting in a host immune response. In addition to forming a physical protective barrier, the gut mucosa also secretes antimicrobials, such as immunoglobulin (Ig) A, antimicrobial substances and reactive oxygen species (ROS), to actively defend the host against invading microbes (Macpherson & Slack 2007, Rios-Arce et al. 2017). The generation of such protective molecules is enhanced by microbiota-derived short-chain fatty acids (SCFAs), strengthening the intestinal epithelial barrier (Kamada et al. 2013, Park et al. 2016). Monocolonization of GF mice with Bacteroides thetaiotaomicron, one of the most abundant anaerobic gut commensals, showed a significant shift in the transcriptional host response by inducing α1,2-fucosyltransferase mRNA expression in the ileum, indicating the recognition of microbiota by the host via pattern-recognition receptors (PRRs) binding to microbe-associated molecular patterns (MAMPs) (Bry et al. 1996, Hooper et al. 1999, 2001, Hooper & Gordon 2001, Ishii et al. 2008, Jones et al. 2012). The basal, low-level PRR activation by gut commensals was shown to have positive influences on normal homeostatic cell renewal and repair processes (Rakoff-Nahoum et al. 2004, Lee et al. 2006, Jones et al. 2012). For example, it was shown that the commensal gut microbiota influences the intestinal barrier integrity via Toll-like receptor (TLR) 2, which results in the suppression of Hh and neuropillin-1 (NRP1) signaling pathways in the IECs, and thus a weakened intestinal barrier (Pontarollo et al. 2023).
Microbiota-induced changes of intestinal vascular morphology - birth and weaning
Besides in the maintenance of the intestinal barrier, the interaction of host and microbiota plays a crucial role in the remodeling of morphology in the small intestine. As a reaction to environmental changes the system is exposed to, the complex and dynamic interaction between the symbionts and their host can adapt and optimize its function. Especially, studies in GF mice have shown the crucial influence of the gut microbiota on physiological processes and the intestinal phenotype of the host (Thompson & Trexler 1971). An important observation in GF mice was the significant enlargement of the cecum (Asano 1969). Due to the absence of microbiota, which are responsible for the digestion of complex carbohydrates and the fermentation of dietary fibre, the undigested components accumulate in the cecal lumen, enlarging its size by 4-8-fold compared to their CONV-R counterparts (Al-Asmakh & Zadjali 2015). Of note, the small intestinal microanatomy was reported to be affected under GF conditions too. Here, villus morphology was strikingly altered in GF mice, as they exhibit a thinner intestinal wall and more narrow, pointier villus structures compared to CONV-R control mice (Thompson & Trexler 1971, Stappenbeck et al. 2002, Reinhardt et al. 2012). Furthermore, GF mice exhibit a thinner small intestinal lamina propria with a reduced mucosal surface area (Gordon & Bruckner-Kardoss 1961). Comprehensive gene expression analyses performed in adult GF mice hinted toward a persisting state of functional immaturity, correlating with the absence of microbiota (Hooper et al. 2001, Stappenbeck et al. 2002). Hence, the spotlight turned on the initial trigger of microbiota-induced remodeling in the small intestine, which, under physiologic conditions, occurs at birth.
After the sterile habitation in utero, the fetus undergoes the passage through the birth canal, where it is initially colonized by microbes (Reinhardt et al. 2009, Bäckhed et al. 2015b). This is followed by a continuous exposure to numerous environmental bacteria colonizing the newborn host on distinct body surfaces (oral cavity, gastrointestinal tract, vagina, skin) (Khandagale & Reinhardt 2018). In the first months of life, the microbial composition undergoes frequent and severe changes, as the infant is exposed to a rapidly changing environment with a broad variety of bacteria (Hooper 2004, Bjursell et al. 2006, Palmer et al. 2007). Mouse studies focusing on the early postnatal development of the small intestine reported that the crypt stem cell hierarchy establishes during the suckling period, which generally lasts for 14 postnatal days (P1–14) (Savage 1977, Schmidt et al. 1988, Wong et al. 2002). Meanwhile, the gut microbiota predominantly consists of facultative anaerobes, such as Proteobacteria (Escherichia coli) and Actinobacteria (Bifidobacterium spp.) (Savage 1977, Mackie et al. 1999). Here, the vascular system of the villus is still rudimental, consisting of a simple arch with only few or no cross-linking branches (Stappenbeck et al. 2002). Within the following 14 days (until P28), when the transition from suckling to weaning takes place, the intestinal microbial composition changes by reducing the abundance of facultative anaerobes and expanding the numbers of obligate anaerobes, predominantly species of Bacteroides, Bifidobacterium, and Clostridium (Moore & Holdeman 1974, Savage 1977, Mackie et al. 1999, Hooper 2003). This pivotal change of the intestinal microflora toward obligate anaerobes provides the basis of a healthy adult microbial flora, ensuring nutrient availability, as young mammals are weaned from the mother’s milk to solid food (Savage 1977, Mackie et al. 1999, Hooper 2003). Along with the dynamic changes of the gut-residing microbiota during the weaning period, the complex villus vasculature is established in its entirety (Stappenbeck et al. 2002). Upon weaning, the small intestine also undergoes a dramatic shift in its metabolic capacity, which simultaneously affects the metabolism of the gut bacteria (Reinhardt et al. 2009). This was shown by global gene expression profiling in gnotobiotic mice, where Bacteroides thetaiotaomicron changes its source for polysaccharides from host derived to plant derived (Bjursell et al. 2006, Reinhardt et al. 2009). Finally, the adult human gut is colonized by an estimate of 150–170 bacterial species, which exert their full symbiotic functions regarding nutrient degradation, nutrition, immune interactions, and morphological and physiological remodeling (Laterza et al. 2016, Adak & Khan 2019, Rinninella et al. 2019).
Small intestinal villus vascularization
The small intestinal blood vasculature
The dietary nutrients are absorbed by the IECs of the small intestine, from where they need to be transported and distributed throughout the whole body. Thus, a complex capillary network in the intestinal villus structures is crucial, not only for supplying the epithelial cells with the nutrients and oxygen but also to transport carbon dioxide to lungs and the absorbed nutrients to the liver via the portal vein and distribute them to metabolically active tissues, comprising the bidirectional gut–liver axis (Albillos et al. 2020). One example is the transport of the intestine-derived high-density lipoprotein cholesterol (HDL-C), which is crucially involved in the absorption of lipids in the circulation, transporting them to the liver where they are metabolized or excreted, comprising an essential role in cholesterol metabolism. Here, recent works of Han and colleagues showed that the intestine-derived HDL reaches the liver via the portal vein, where it was shown to bind gut-derived proinflammatory LPS, preventing LPS-induced liver injury (Han et al. 2021). These findings substantiate the anti-inflammatory and antimicrobial effects of HDL, synthesized in the intestine and the importance of the gut–liver axis (Han et al. 2021, Kim & Seki 2022). To provide the best possible exchange with the epithelial cell lining, the underlying blood vessels are built in a complex cage-like structure (Fig. 2A). The inner lining of this intestinal blood microvasculature is composed of endothelial cells, which compose a selectively permeable barrier (Fig. 2A). This endothelial barrier allows the selective passage of small molecules including oxygen, carbon dioxide, and nutrients to maintain the supply of the surrounding tissue, while large molecules and cells are typically prevented from freely crossing the barrier (Claesson-Welsh et al. 2021). Besides the selective barrier function, endothelial cells are known to exert a multitude of functions by participating in the inflammatory response, coagulation, thrombosis, regulation of the vascular tone, and the secretion of regulatory molecules. In addition, vascular endothelial cells are known to initiate angiogenesis in response to tissue injury or hypoxic conditions (Carmeliet & Jain 2011).
Schematic view of the microvasculature and its cross section found within each small intestinal villus. (A) The blood capillaries are lined by endothelial cells, surrounded by the basal lamina containing pericytes. (B) The lacteal vessel is lined by endothelial cells connected by specialized junctions allowing chylomicron passage. Both vessel systems are surrounded by smooth muscle cells. Created with BioRender.com.
Citation: Microbiota and Host 2, 1; 10.1530/MAH-23-0021
Located underneath the vessel wall of endothelial cells, the basal lamina provides structural support to the vessel (Fig. 2A). This layer harbors pericytes, forming an envelope around the surface of the vessel tube staying in close contact to the endothelial cells. Pericytes are known to regulate blood flow, contribute to the stabilization of the capillary wall, and are involved in angiogenesis (Gerhardt & Betsholtz 2003, Armulik et al. 2005, von Tell et al. 2006, Ribatti et al. 2011, Stapor et al. 2014). This intricate system of blood vasculature is embedded in the lamina propria and projects into the center of each villus, extending toward the tip and forming a network of anastomosing capillaries (Wilting & Christ 1996, Ramirez et al. 2019). It is surrounded by SMCs, which provide stability and regulatory signals for the vasculature network.
The small intestinal lymphatic vasculature
Enclosed by the blood capillary cage, a blunt-ended lacteal can be found in the center of each small intestinal villus (Alexander et al. 2010, Miller et al. 2010, Bernier-Latmani & Petrova 2017) (Fig. 2B). The lacteal connects to the lymphatic system passing through the lamina propria, providing lymphatic flow into the villus transporting immune cells. This represents an essential part of the gut immune surveillance system, establishing a tight regulation between the tolerance of harmless commensals as well as dietary antigens and the protection against pathogens (Van Kruiningen & Colombel 2008, Miller et al. 2010, Vetrano et al. 2010, Dieterich et al. 2014, Bernier-Latmani & Petrova 2017, Suh et al. 2019). The lacteal is lined with specialized lymphatic endothelial cells (LECs), which are interconnected by two different types of junctions, zipper-like junctions and button-like junctions, maintaining the lacteal integrity, with button-like junctions being primarily found in initial lymphatic vessels while zipper-like functions dominating the collecting lymphatic vessels (Baluk et al. 2007). Both integrity and maintenance of the lacteal are further influenced by continuous signals provided by adjacent stromal cells (Bernier-Latmani & Petrova 2017) such as SMCs, periodically squeezing the lymphatic endothelial tube supporting the lymphatic drainage (Choe et al. 2015) and releasing vascular endothelial growth factors (VEGFs), which activate their respective receptors (VEGFRs) expressed by LECs (Nurmi et al. 2015). One of the major tasks of the lacteals is the transport of dietary fats, which are absorbed and resynthesized into triglycerides by enterocytes (Dixon 2010, Ko et al. 2020). The newly formed triglycerides are packaged into lipoprotein particles, termed chylomicrons, and enter the lacteal through the LECs into the lacteal lumen and travel via the lymphatic vasculature, as they cannot enter the blood stream directly due to their large size. The lacteal of the small intestinal villus merges with larger lymphatic vessels, which ultimately empty into the thoracic duct, where the transported chylomicrons enter the venous circulation (Miller et al. 2010, Bernier-Latmani & Petrova 2017). Herein, the fatty acids are released from the chylomicrons and are available for peripheral tissues. The scavenger HDLs can then collect excess lipids, mostly in the form of cholesterol, to transport them to the liver for their final metabolic degradation (Dixon 2010, Randolph & Miller 2014, Bernier-Latmani & Petrova 2017).
Of note, the barrier composed of endothelial cells can be seen as a secondary intestinal barrier, which in addition to the first epithelial monolayer barrier protects against pathogen invasion from the gut (Jang et al. 2013, Suh et al. 2019, Albillos et al. 2020). Similar to blood vessels, the LEC junctions prevent lymph leakage into the surrounding tissue on the one hand while allowing the passage of certain absorbed dietary components for further transport on the other hand (Baluk et al. 2007, Kim et al. 2007, Zheng et al. 2014, Xia et al. 2022). Tight regulation of the gut barrier is crucial, since the dysfunction of intestinal lymphatic vasculature has been associated with inflammatory bowel disease (Van Kruiningen & Colombel 2008, Tonelli et al. 2012, Rahier et al. 2013). Here, the protective role of lymphatic vessels was demonstrated, as the blockade of VEGFR3, a crucial growth factor for lymphatics, resulted in an aggravation of intestinal inflammation (Jurisic et al. 2013), while VEGF-C stimulation was shown to improve lymphatic function resulting in the amelioration of colitis symptoms (D'Alessio et al. 2014).
The role of the microbiota in small intestinal vascularization
The essential process of vasculogenesis, which is the de novo formation of vessels, is predominantly initiated by the intrinsic factors released during embryogenesis and lays the foundation for the primary blood and lymphatic vascular network before angiogenesis takes over. The development of the circulatory system in the small intestine during embryogenesis is reviewed in more detail elsewhere (Henderson et al. 2020, Bayer et al. 2021). Here, several growth factors and their respective receptors have been determined to drive vasculogenesis, for example the VEGFs binding to their respective VEGFRs, which leads to the assembly and differentiation of angioblasts and endothelial progenitor cells to form new vessels (Kubis & Levy 2003, Henderson et al. 2020, Bayer et al. 2021). It has been shown that LECs comprising the small intestinal lacteals, in contrast to lymphatic vessels in other organs, sustain their regenerative and proliferative capacity under steady-state conditions, requiring various regulators. Among those mediating pathways, VEGF signaling and continuous activation of Notch ligand delta-like 4 (DLL4) signaling can be found, enabling the lacteals to remodel and adapt in response to environmental factors even in adulthood (Bernier-Latmani et al. 2015).
While the vasculogenesis primarily takes place during embryonic development, the process of angiogenesis, which is the formation of new vessels from preexisting ones, dominates postnatal development in occasions such as wound healing, tissue repair, and tumor growth. Here, pro- and anti-angiogenic factors are essential for the tight regulation of angiogenesis in order to maintain tissue health and function.
Besides the beneficial physiological functions, e.g. restoring oxygen and nutrient supply via blood vessel formation in injured/traumatized tissue (wound healing), angiogenesis can also have pathological roles such as growing tumors utilizing angiogenesis to ensure the supply of oxygen and nutrients. Many studies have revealed various physiological and pathological factors influencing the initiation of the angiogenic program in target tissues. Among others, coagulation is a key process initiating angiogenesis by releasing various growth factors and signaling molecules during the clotting process, as it is found, for example, in wound healing. Here, coagulation is the body’s natural response to injury by assembling blood components, such as platelets and clotting factors (e.g. tissue factor (TF) or factor X), to form a blood clot and prevent excessive bleeding. These coagulation factors can simultaneously act as signaling molecules, stimulating endothelial cells lining the blood vessels to proliferate and migrate, ultimately leading to the extension of the existing vessel. The development of lacteals occurs during embryogenesis and is continued during postnatal development (Kim et al. 2007), especially maturing the length and junctional patterns of the lacteals (Suh et al. 2019). Of note, the pivotal changes in the gut microbiome occurring during the weaning period were also shown to affect the processes involved in lacteal maturation (Suh et al. 2019).
The genetic program applied during postnatal vascular development in the gut is crucially affected by the presence of microbiota (Stappenbeck et al. 2002, Reinhardt et al. 2012). However, the mechanisms by which gut commensals impact small intestinal vascularization are limitedly understood. Given the pivotal role of gut capillaries in nutrient harvest, the comprehensive analysis of gut microbiota affecting small intestinal vascularization aroused significant interest.
Intriguingly, CONV-D mice (ex-germ-free mice colonized with a complex gut microbiota) showed a complete restoration of the villus vasculature indistinguishable from CONV-R within 10 days, suggesting a link between vascular remodeling and alterations in villus architecture upon microbial colonization (Stappenbeck et al. 2002, Reinhardt et al. 2012). Additionally, monoassociation experiments with Bacteroides thetaiotaomicron showed that a single bacterial strain, which is a prevalent intestinal resident in humans and mice, was able to induce the remodeling of villus vascularization up to CONV-R levels (Moore & Holdeman 1974, Stappenbeck et al. 2002, Reinhardt et al. 2012). Furthermore, recent studies have shown that the consortium of 14 selected bacterial strains of the Oligo-Mouse-Microbiota (OMM) community (Brugiroux et al. 2016) not only stably colonizes the gut of adult GF mice, leading to stimulation of morphological and immunological maturation, but also triggers the development of the villus capillary network in the intestine (Romero et al. 2022). Intriguingly, the enrichment of the OMM community with E. coli and Citrobacter amalonaticus colonizing the gut of GF mice resulted in the transcriptional upregulation of angiopoietin-like 4, vasohibin 2, and the adrenomedullin-receptor activity-modifying protein 2 system in epithelial and endothelial cells, which are known to be involved in blood vessel development, vascular integrity, and homeostasis (Babapoor-Farrokhran et al. 2015, Ochoa-Callejero et al. 2016, Romero et al. 2022). These findings support the assumption of the gut abiding in a state of functional immaturity until adulthood among the absence of microbiota (Stappenbeck et al. 2002). Thus, the adult intestine maintains the developmental advantage of rapid dynamics and adaptability, enabling the spontaneous reaction to external influences, such as microbial induction of angiogenesis.
Vascular remodeling induced by the gut microbiota – proposed pathways
Although small intestinal villus vascularization is strongly associated with changes in the microbial community of the gut, only little is known about the exact mechanisms involved in the remodeling of this vascular network. Thus, different pathways were proposed, which potentially mediate microbiota-induced angiogenesis, and will be discussed here.
One proposed mediator of microbiota-induced angiogenesis is the Paneth cells (Fig. 3A). Cells differentiate and reside in the villus crypts of the intestine (Cheng & Leblond 1974, Bjerknes & Cheng 1981), where they comprise a key component of the intestinal innate immunity. Through antimicrobial peptide secretion into the gut lumen, Paneth cells are known to signal and interact with the gut-residing microbiota (Ouellette & Selsted 1996, Ayabe et al. 2000). It was reported that mice lacking Paneth cells from their intestinal crypts showed less complex capillary networks compared to their wild-type littermates, both in GF and CONV-R states (Stappenbeck et al. 2002). This work indicates that Paneth cells are involved in the development of the villus microvasculature, even in the absence of microbiota (Stappenbeck et al. 2002). This is in line with the finding that at P28, the full and complex establishment of the villus vascular network coincides with the appearance of differentiated Paneth cells in the now fully formatted crypts, which considers them as important contributors to small intestinal angiogenesis. In contrast, during the first 2 weeks of postnatal development, the microvascular pattern is still very simple consisting of a single arch with only few or no cross-linking branches (Stappenbeck et al. 2002), while mature Paneth cells are also missing from the gut epithelium (Bry et al. 1994). Furthermore, the absence of Paneth cells in GF mice showed no remodeling of villus capillaries upon monocolonization with B. thetaiotaomicron, in contrast to wild-type mice, underscoring the crucial role of Paneth cells in villus vascular remodeling (Stappenbeck et al. 2002). Supporting this notion, the Paneth cell granule protein angiogenin-4 (ANG4) expression was found to be upregulated upon weaning, where it can exert its microbicidal functions (Hooper et al. 2003). While Ang4 expression was shown to depend on microbial signals from gut commensals, it provides a potential target of a vascular remodeling factor induced by microbiota (Strydom 1998). Experiments by Hooper and coworkers have shown that Ang4 expression was significantly increased in CONV-R mice, compared to GF mice (Hooper et al. 2003). The influence of microbiota on Ang4 mRNA expression could be confirmed by colonizing GF mice with a CONV-R-intestine-derived microbiota resulting in the restoration of Ang4 expression, while even the monocolonization of GF mice with B. thetaiotaomicron was sufficient to induce adult levels of Ang4 (Hooper et al. 2003). However, the microbiota-dependent signaling mechanism of Ang4 on villus vascularization is yet to be determined.
Proposed pathways of microbiota-mediated vascular remodeling. Schematic view of microbiota-mediated vascular remodeling (A) via Paneth cells signaling (Stappenbeck et al. 2012), (B) via an epithelial–endothelial cross talk with microbiota affecting TF glycosylation leading to PAR1 activation, which initiates angiogenesis via the Ang-1/TIE2 pathway (Reinhardt et al. 2012), and (C) via TLR/TIR activation by microbiota via NFκB, VEGF-R2, and FAK involving TRAF6 and MAPK signaling, leading to promotion of angiogenesis. (D) TLR activation triggering MyD88 signaling, leading to an increased VEGF-C expression in villus macrophages, which binds to VEGFR3 on LECs, affecting lacteal integrity (Suh et al. 2019). Created with BioRender.com.
Citation: Microbiota and Host 2, 1; 10.1530/MAH-23-0021
An additional pathway proposed by Reinhardt et al. recapitulated the findings of increased villus vessel density upon microbial colonization by Stappenbeck et al., showing increased expression of vascular and angiogenic markers in the small intestine of colonized mice. In contrast, they identified a potential epithelial–endothelial cross talk mediating microbiota-regulated villus vascularization, which is independent of Paneth cells. In this study it was revealed that the glycoprotein angiopoietin-1 (ANG-1), a member of the angiopoietin family of growth factors, which are known to function in later stages of vascular development, remodeling, and stabilization of adult vessels, appears to be essential for microbiota-induced vascular remodeling (Brindle et al. 2006, Reinhardt et al. 2012). Furthermore, the analysis of TF expressed on small intestinal enterocytes, with TF being a prominent initiator of the coagulation protease cascade generating proteolytic fragments with potent regulatory effects on angiogenesis, showed that gut microbiota promotes N-glycosylation of TF, affecting its localization to the cell surface, which affects its activity in coagulation factor signaling (Reinhardt et al. 2012) (Fig. 3B). Due to its activating role of coagulation proteases, TF is a known initiator of angiogenesis. Therefore, the microbiota-triggered modification of TF on IECs, resulting in altered procoagulation activity, may impact angiogenesis in small intestinal tissue. Further, experiments conducted on protease-activated receptor (PAR)1-deficient mice showed that the gut microbiota is able to induce PAR1 expression, which was attributed to vascular remodeling in the small intestine. In summary, this study showed that TF and PAR1 promote a microbiota-induced intestinal vascular remodeling, with TF being expressed on the basolateral site of intestinal epithelial cells and being subjected to glycosylation promoted by the residing microbiota (Camerer et al. 1996). The glycosylated TF then initiates extravascular coagulation signaling pathways via PAR1. This augments the expression and signaling of ANG-1, finally leading to vascular remodeling and angiogenesis in the small intestine (Reinhardt et al. 2012) (Fig. 3B). Intriguingly, recent findings reported the ectopic, microbiota-dependent generation of thrombin, another crucial hemostatic factor in coagulation, by gut epithelial cells (Motta et al. 2019). Here, it was shown that active thrombin is released at the luminal site of the intestinal mucosa of CONV-R mice, while GF mice exhibit a reduction of colonic thrombin expression, suggesting the microbiota as a controlling factor of intestinal epithelial thrombin generation (Motta et al. 2019). Traditionally, the serine protease thrombin is known to promote platelet aggregation via PAR activation during hemostasis. Interestingly, Motta and coworkers demonstrated that epithelial thrombin activity preserves intestinal mucosal homeostasis, e.g. by regulating the microbial community composition and the integrity of the mucosal biofilm, preventing microbial invasion (Motta et al. 2019).
As a third potential pathway of microbiota influencing intestinal vascularization, the signaling via the innate immune pattern-recognition receptors, Toll-like receptors (TLRs), was investigated. In particular, it was proposed that besides the ability of commensal microbiota inducing host innate immune responses, it can also activate microvascular cells in order to induce angiogenesis (Schirbel et al. 2013). Here, the effect of bacterial ligands binding TLR2/6 and TLR4 in human intestinal microvascular endothelial cells and isolated fibroblasts was assessed. It was demonstrated that the gut microbiota-derived signals were able to activate TLR signaling ultimately resulting in the promotion of angiogenesis indicated by an increased proliferation, migration, tube formation, and vessel sprouting of endothelial and mesenchymal cells (Schirbel et al. 2013). It was shown that the innate immunity-mediated response to the microbial signals was conveyed by tumor necrosis factor receptor-associated factor 6 (TRAF6)-dependent signaling involving the mitogen-activated protein kinase (MAPK) and nuclear factor kappa-light chain enhancer of activated B cells (NFκB) pathways as well as VEGFR2 and focal adhesion kinase (FAK), ultimately promoting angiogenesis (Schirbel et al. 2013). In line with these findings, it was shown that the postnatal development of the vascular endothelium in the small intestine was strongly dependent on the presence of TLR/interleukin-1 receptor (TIR), an intracellular signaling domain activated by indigenous bacteria (Rakoff-Nahoum et al. 2015). More recently, Schirbel et al. determined the carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) was also subject to microbially activated TLR2/4 and TLR4, among other innate immune receptors, leading to the selective and differential upregulation of cellular and soluble CEACAM1 (Schirbel et al. 2019). Further, it could be shown that CEACAM1, which is known for its function in cell activation, proliferation, pathogen binding, and angiogenesis (Tchoupa et al. 2014), exerts pro-angiogenic effects on intestinal endothelial cells after being induced by bacterial ligand-activated TLR signaling. Thus, these studies substantiate the potential of innate immune receptors, such as TLRs, involving additional mediator molecules, to regulate intestinal vascularization in a gut microbiota-dependent fashion (Fig. 3C).
Lastly, the microbiome has also been reported to be involved in maintaining lacteal integrity. Antibiotic treatment of mice, depleting the majority of the gut microbiome, was shown to trigger lacteal regression during adulthood and delay lacteal maturation during the postnatal period (Suh et al. 2019). This observation was associated with a significant reduction of the proportion of button-like junctions and an increase of the proportion of zipper-like junctions between the LECs, resulting in a compromised lipid absorption in antibiotic-treated mice (Suh et al. 2019). Interestingly, GF mice showed lacteal defects comparable to those observed in antibiotic-treated mice, while conventionalization was sufficient to restore lacteal integrity comparable to CONV-R levels (Suh et al. 2019). Hence, the gut microbiota could be determined as a crucial regulator of the structural and functional integrity of lacteals (Suh et al. 2019). Here, signaling via VEGFR3 was proposed to mediate the microbial effect on lacteal integrity, as mice lacking VEGFR3 in LECs showed similar lacteal phenotypes as GF mice (Suh et al. 2019). In addition, Suh et al. was able to determine villus-resident macrophages as an indispensable source for VEGF-C modulating lacteal integrity dependent on the presence of gut microbiota (Fig. 3D). Based on this study, it was proposed that the adaptor molecule myeloid differentiation primary-response protein 88 (MyD88), which transmits downstream signals of most TLRs, is crucially involved in VEGF-C expression by macrophages in order to maintain lacteal integrity (Fig. 3D). However, it remains unclear which intestinal cell type instructs the TLR-MyD88-mediated microbiota-sensing promoting VEGF-C production by villus macrophages, ultimately influencing lacteal integrity (Suh et al. 2019). Interestingly, in this study, the tissue mRNA levels of additional lymphangiogenic factors, including ANG-1 (Kajiya et al. 2012), ANG-2 (Zheng et al. 2014), transforming growth factor (TGF) β1 (Clavin et al. 2008), collagen- and calcium-binding EGF domains (Bos et al. 2011), and a disintegrin and metalloprotease with thrombospondin motifs 3 (Bui et al. 2016), were not altered in respect to the presence of microbiota (Suh et al. 2019). As already mentioned, the ANG-1/TIE-2 signaling is an essential driver of blood vessel formation. In detail, the interaction of ANG-1 with its receptor TIE-2 is known to maintain angiogenesis and remodeling during the embryonic period, while also promoting lymphatic sprouting and growth (Tammela et al. 2005). Furthermore, ANG-2, which is an antagonist of ANG-1/TIE-2 signaling, was reported to regulate the integrity of intercellular junctions between LECs during lymphatic development, transforming zipper-like junctions into button-like junctions, which ultimately facilitates the fluidic drainage from the initial lacteals to the collecting lymphatic vessels (Zheng et al. 2014).
Overall, the current state of knowledge comprehends a multiplicity of potential factors that are involved in intestinal villus angiogenesis and vascular remodeling in a microbiota-dependent manner. However, the precise interaction between these factors, different cell types, and the microbial commensals remains to be elucidated in future studies.
Conclusion and perspectives
It becomes increasingly clear that the gut microbiota plays a significant role in the regulation of small intestinal villus vascularization. The findings recapitulated here support the assumption of the gut abiding in a state of functional immaturity until adulthood in the absence of microbiota. In general, bacterial interactions with Paneth cells are an important factor of postnatal gut development, including the induction of mucosal antimicrobial defense mechanisms, innate immunity, and potentially also mediating microbiota-induced vascular remodeling. However, it remains elusive which specific bacterial strains are involved in triggering vascular remodeling pathways, as current research based on gnotobiotic mouse models is still limited. Even though big advances in gut microbiome research were achieved, especially with novel techniques like metagenomic sequencing, transcriptome, proteome, and metabolome analysis, gnotobiotic in vivo studies still rely on bacterial strains and isogenic mutant strains that can be cultivated. This remains a current limitation of gut microbiota research, restricting the comprehensive investigation of the crucial (bacterial) players involved in vascular remodeling.
Although the TF–PAR1–ANG-1 interaction presents a promising pathway mediating the microbiota’s impact on villus vascularization, there are many other surface molecules on villus enterocytes, which are in close contact to the microbiota, potentially mediating an epithelial–endothelial cross talk. Microbiota-induced alterations in epithelial molecules, primarily the microbiota-sensing PRRs, might induce downstream signaling cascades ultimately stimulating angiogenesis and vascular remodeling in the small intestine. However, those molecules, which are potentially involved in a microbiota-driven epithelium-to-endothelium cross talk, are still to be determined and investigated. Of note, it is important to determine whether the microbiota acts directly on the epithelial signaling molecules or if microbial signals, e.g. microbial metabolites, are involved in the signal transmission.
Finally, the comprehensive study of microbiota-induced vascular remodeling will not only aid in the fundamental understanding of the symbiotic relationship occurring in the gut but also serve the exploration of potential therapeutic targets. Angiogenesis has already been targeted in the treatment of various diseases such as cancer, retinal diseases, inflammatory disorders, and cardiovascular diseases (Gariano & Gardner 2005, Khurana et al. 2005, Szekanecz & Koch 2007, Fallah et al. 2019, Rahat et al. 2020). For example, external intervention in angiogenesis is aiming to block the blood vessel growth in tumors and thereby limit their access to nutrients, finally resulting in slowing down their growth (El-Kenawi & El-Remessy 2013). Promising studies investigated how lacteal growth could be prevented by deletion of VEGF-C and DLL4. This deletion was reported to leave the mice resistant to high-fat-diet-induced obesity, showing that lacteals can be targeted to prevent obesity (Bernier-Latmani et al. 2015, Nurmi et al. 2015). Furthermore, molecules involved in the junction transformation of lacteals in obesity comprise a promising target for obesity treatment by limiting the transition of dietary fats from the intestine to the circulation (Ko et al. 2020). Overall, also the targeted alteration of the small intestinal gut microbiota in the small intestine may be a potent way to specifically regulate host metabolism and the response to dietary lipids (Ko et al. 2020).
In summary, the intricate interplay between the gut microbiota and its host has long been a subject of scientific curiosity and has unraveled the fascinating impact commensals have on the small intestinal physiology, including nutrient uptake, immune surveillance, villus morphology, and mucosal vascularization. Thus, gut commensals have emerged as a novel target in angiogenesis regulation. Understanding the pathways regulated by the microbiota will provide a potent tool to influence host physiology and morphology, especially the small intestinal vascularization.
Declaration of interest
The authors declare that there are no conflicts of interest that could be perceived as prejudicing the impartiality of this work.
Funding
This work was supported by the BMBF Cluster4Future CurATime (MicrobAIome, grant number 03ZU1202CA, to CR), the Deutsche Zentren der Gesundheitsforschung (DZG) Innovation Fund “Microbiome” (81X2210129 to CR), a project grant by the Wilhelm Sander-Stiftung (2022.131.1 to CR), and the Forschungsinitiative Rheinland-Pfalz and ReALity. CR was awarded a fellowship from the Gutenberg Research College at Johannes-Gutenberg University Mainz.
Author contribution statement
NP conceived the scope of the paper, reviewed literature, and wrote and edited the manuscript. CR conceptualized the manuscript, reviewed literature, wrote and edited the manuscript. All authors approved the manuscript prior to submission.
Acknowledgements
CR is a member of the Center for Translational Vascular Biology (CTVB), the Research Center for Immunotherapy (FZI) at the University Medical Center Mainz and the Potentialbereich EXPOHEALTH at Johannes Gutenberg-Univertsity Mainz. CR is a scientist at the German Center for Cardiovascular Research (DZHK) and a PI at the BMBF Cluster4Future CurATime. NP is a PhD student at the Mainz Research School of Translational Biomedicine.
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