Cellular pathogenesis of gastroparesis

Abbreviations

Introduction

Delayed emptying of gastric contents in the absence of any mechanical obstruction of the gastric outlet is pathognomonic for gastroparesis . As opposed to normal GI function, delay in gastric emptying may be due to antral dysmotility , impaired fundic accommodation , and/or pyloric dysfunction . The cardinal symptoms of this chronic disorder include early satiety, postprandial fullness, abdominal pain, nausea and vomiting . These result in significant impairment of quality of life and high healthcare expenditures .

Although, diabetes has been considered to be the leading cause of gastroparesis, a long-term follow-up study accounted for only 29% patients harboring diabetic gastroparesis (DG), while gastroparesis acquired after a surgery (post-surgical gastroparesis) constituted 13% of the cases . A majority of patients (>50%) presented with an unknown primary cause and were categorized as idiopathic gastroparesis (IG) . However, a recently published cross-sectional study in a large and diverse US population, only 28% of patients represented IG whereas, 72% patients exhibited DG (16% type 1 diabetes mellitus and 55% type 2 diabetes mellitus) . These differences may be attributed to the diagnostic code-based approach for the identification of gastroparesis in the recent compared to gastric scintigraphy confirmed gastroparesis in the prior study. Other commonly observed etiologies of gastroparesis include connective tissue disorders, Parkinson’s disease, or medication-induced .

Gastric emptying is considered to be abnormal/delayed when >60% of administered contents are retained at 2 hours and/or >10% at 4 hours on a solid meal gastric scintigraphy . The severity of symptoms associated with gastroparesis is assessed using the Gastroparesis Cardinal Symptom Index (GCSI) which is based on three subscales: early satiety/postprandial fullness, nausea/vomiting, and bloating . Even though the abdominal pain is not included in the list of GCSI subscales, pain is observed in >80% patients with gastroparesis . Interestingly, a very recent report by Hasler et al. indicates the use of opioids is common in both DG and IG patients and associates with worse cardinal symptoms of gastroparesis and increased hospitalization .

Decades of research in the field of gastroparesis using complimentary animal models and human biopsy specimens, has significantly helped in understanding the cellular and molecular changes in disease pathogenesis. In this chapter, we aim to highlight the research advancements in understanding the gastroparesis pathophysiology and strategies for minimally invasive procurement and use of human full thickness biopsy specimens for future research.

Complexity of gastric emptying

Emptying of gastric contents into the small bowel is a highly complex physiological process involving the synergetic interactions of the neuro-muscular apparatus of the stomach. Normally, emptying of gastric contents occurs by coordinated and appropriate movements of the fundus, body, antrum, and pylorus. These movements occur by the critical roles played by extrinsic nerve innervations, excitatory and inhibitory components of the enteric (intrinsic) nervous system (ENS), interstitial cells of Cajal (ICC), fibroblast-like cells and smooth muscle cells (SMC) . These different cell types are located deep within the muscular and myenteric layers of the stomach. In addition, different parts of the stomach and the feedback loop between the stomach and small intestine also play important roles in mediating gastric emptying . For instance, the fundus and the upper body portions of the stomach generate a basal pressure by producing low frequency, sustained contractions. Similarly, the lower body portion and the antrum of the stomach generate contractions by producing strong peristaltic waves. These tonic contractions assist in pushing gastric contents towards the pylorus while, the digestible solids larger than 2 mm are bounced backwards. This process of mixing and churning occurring due to phasic contractions completes the process of homogenizing the ingested food. The digested food is then emptied into the small bowel as the increased amplitude of peristaltic waves obliterates the lumen of the pylorus, delivering the gastric contents in small volumes of liquid and chyme .

Gastric motility is controlled by a set of neural and hormonal signals. GI tract stands distinctive by having its own intrinsic neuroglial circuits (the ENS). Excitatory and inhibitory neurons are categorized based on the expression pattern of specific neurotransmitters. For example, excitatory neurons express choline acetyltransferase (ChAT) , substance P (SP) and neurokinins (NKA, NKB & neuropeptide Y) whereas, the inhibitory neurons express neuronal nitric oxide synthase (nNOS) - which generates nitric oxide (NO) , and vasoactive intestinal polypeptide (VIP) . In addition, specialized functions like fundic accommodation and pyloric relaxation are mediated by inhibitory nitrergic nerves .

ICC are the mesenchymal cells located within the musculature of the GI tract and are well known for their intrinsic ‘pacemaker’ activity . They play a vital role in generating bioelectrical slow wave potential in a spatiotemporal manner , driving rhythmic contraction of SMC . The syncytial network of ICC is known to mediate sequential excitatory and inhibitory neuro transmissions and also does play a role in mechanotransduction – the critical components of normal gastrointestinal motility . Populations of ICC are recognized typically by their surface expression of receptor tyrosine kinase (Kit) and anoctamin-1 (Ano-1) – a calcium activated chloride channel . NO has been shown to be a survival factor for ICC in mouse models and NO also promotes ICC proliferation in vitro , indicating the association of nitrergic neurons with ICC. Changes in the expression and splicing of Ano-1 have been associated with symptoms of DG .

Fibroblast-like cells are a second population of interstitial cells located in the tunica muscularis of the GI tract and impart additional regulatory control in the ENS . These cells are shown to express cell surface receptor tyrosine kinase - platelet-derived growth factor alpha (PDGFRα) and not Kit , hence are popularly known as PDGFRα ⁺ cells. Ultrastructure studies have shown fibroblast-like cells exhibiting features of fibroblasts and also have connections with circular and longitudinal SMC via gap junctions . In addition, these fibroblast-like cells are also shown to be closely associated with intramuscular ICC and enteric nerve fibers . The ICC, fibroblast-like cells and SMC together form an electrically coupled unit termed SIP syncytium – an important unit for normal GI motility .

Smooth muscle of GI tract is required to crush, grind and mix the intraluminal contents, liquefying them to form chyme. The chyme is then pushed through the pyloric canal into the duodenum establishing gastric emptying. This process of gastric emptying is accomplished by phasic contractions of SMC . The SMC themselves do not originate slow waves – a rate limiting step of gastric emptying. Instead, the SMC are linked to the neighboring ICC via gap junctions creating a syncytium , which drives coordinated contraction and relaxation of SMC by producing bioelectrical slow waves . These phasic contractions and relaxations of the SMC are also regulated by NO from nitrergic neurons and the mechanism is mediated by NO-sensitive guanylyl cyclase (NO-GC) .

Macrophages play a pivotal role in maintaining tissue homeostasis by responding to internal and external stimuli. Resident macrophages are highly heterogeneous in nature and the tissue microenvironment guides the macrophages to attain a specific phenotype. Each of these phenotypes is known to have variable gene expression profiles . Variability is often seen in the expression pattern of well-established macrophage markers between mouse and humans ; for example, iNOS, Arginase-1 and Ym1 are predominantly expressed by mouse but not by human macrophages . Indeed, variability in macrophage gene expression profiles can also be observed when compared not only between the species but, also within a tissue from the same host. For example, the gene expression profile of macrophages in the GI tract varies from mucosa, submucosa, muscularis and serosa in mouse . Typically, muscularis macrophages of mouse predominantly express CX3CR1 (hi), MHCII (hi) and CD11c (lo), whereas, lamina propria macrophages express CD11c (hi) . Likewise, high levels of CD11b and CD14 are expressed by human muscularis macrophages but, a reduced level of their expression is seen in mucosal macrophages . Phenotypically, macrophages located in the circular muscle and longitudinal muscle are of bipolar shape, whereas, myenteric macrophages are stellate shaped .

In the context of GI tract, the studies concerning macrophages are mostly focused on the lamina propria macrophages. Wherein, these macrophages play defensive roles by acting against harmful gut microbial invasion, engulfing and clearing luminal antigens that occasionally breach mucosal barrier. Recently, muscularis macrophages are gaining attention of the researchers as they stand unique transcriptionally and also morphologically from the neighboring macrophage counterparts of mucosa and lamina propria . There is a close and likely symbiotic interaction between the macrophages and ENS in the myenteric plexus . Macrophages secrete a protein belonging to TGF-β superfamily called bone morphogenetic protein-2 (BMP-2) . Enteric neurons express BMP receptor (BMPR) and binding of BMP-2 to BMPR leads to oligomerization of type I and type II serine kinases, followed by phosphorylation and nuclear translocation of SMAD proteins . Activation of BMPR promotes nitrergic enteric neuronal differentiation and helps in regulating gastrointestinal motility . And also, BMP-2 has been shown to promote nNOS expression in myenteric neurons through SMAD1 phosphorylation . In turn, enteric neurons produce colony stimulating fator-1 (CSF-1) – a growth factor essential for the differentiation and maintenance of muscularis macrophages and this is evident from the osteopetrotic ( op/op ) mice, which harbor a mutation in their CSF-1 gene, also lack muscularis macrophages and display a disordered ENS architecture . In addition, ICC has been identified as a non-neuronal source of CSF-1 in mice lacking enteric neurons (RetKO mice) . A recent report by Cipriani et al. shows an increased number of neurons in Csf1 ^op/op mice expressing an overlap between NOS1 and ChAT–a chemical code overlap that is known to reduce as the neurons mature . Thus, cross-talk between ENS, ICC, fibroblasts like cells, SMC and immune cells play an indispensable role in maintaining normal gastric physiology.

Pathogenesis of gastroparesis is associated with cellular alterations

Research so far in delineating the pathophysiological alterations associated with gastroparesis has been successful in finding several key anomalies associated with some of the important cell types ( Fig. 6.1 ). In addition, very recently, transcriptomic and proteomic analyses have been successful in identifying the molecular changes occurring during the pathogenesis of gastroparesis. Here we describe some of the important studies that have impacted our understanding of the pathological and molecular basis of gastroparesis.