Evidence of enteric angiopathy and neuromuscular hypoxia in patients with mitochondrial neurogastrointestinal encephalomyopathy

Abstract

Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is a rare autosomal recessive disease caused by thymidine phosphorylase (TP) enzyme defect. As gastrointestinal changes do not revert in patients undergone TP replacement therapy, one can postulate that other unexplored mechanisms contribute to MNGIE pathophysiology. Hence, we focused on the local TP angiogenic potential that has never been considered in MNGIE. In this study, we investigated the enteric submucosal microvasculature and the effect of hypoxia on fibrosis and enteric neurons density in jejunal full-thickness biopsies collected from patients with MNGIE. Orcein staining was used to count blood vessels based on their size. Fibrosis was assessed using the Sirius Red and Fast Green method. Hypoxia and neoangiogenesis were determined via hypoxia-inducible-factor-1α (HIF-1α) and vascular endothelial cell growth factor (VEGF) protein expression, respectively. Neuron-specific enolase was used to label enteric neurons. Compared with controls, patients with MNGIE showed a decreased area of vascular tissue, but a twofold increase of submucosal vessels/mm² with increased small size and decreased medium and large size vessels. VEGF positive vessels, fibrosis index, and HIF-1α protein expression were increased, whereas there was a diminished thickness of the longitudinal muscle layer with an increased interganglionic distance and reduced number of myenteric neurons. We demonstrated the occurrence of an angiopathy in the GI tract of patients with MNGIE. Neoangiogenetic changes, as detected by the abundance of small size vessels in the jejunal submucosa, along with hypoxia provide a morphological basis to explain neuromuscular alterations, vasculature breakdown, and ischemic abnormalities in MNGIE.NEW & NOTEWORTHY Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is characterized by a genetically driven defect of thymidine phosphorylase, a multitask enzyme playing a role also in angiogenesis. Indeed, major gastrointestinal bleedings are life-threatening complications of MNGIE. Thus, we focused on jejunal submucosal vasculature and showed intestinal microangiopathy as a novel feature occurring in this disease. Notably, vascular changes were associated with neuromuscular abnormalities, which may explain gut dysfunction and help to develop future therapeutic approaches in MNGIE.

Keywords: angiogenesis; fibrosis; gastrointestinal bleeding; platelet-derived endothelial cell growth factor 1; submucosal vessels.

Graphical abstract

Figure 1.

Measurement of vascular tissue of the submucosal layer. A: ratio of the area of submucosal layer occupied by vascular tissue expressed in mm² divided for the total area (mm²) of submucosa in patients with MNGIE and controls (CTR) (*P = 0.0022). B: ratio between the number of total vessels counted in the submucosa divided for the total area (mm²) of submucosa in patients with MNGIE and CTR (*P = 0.019). C: illustration of the percentage of each group of vessels separated on the basis of their diameter in the submucosa of patients with MNGIE and CTR. Note that in patients with MNGIE more than half of the vessels have the diameter < 50 µm. MNGIE, mitochondrial neurogastrointestinal encephalomyopathy.

Figure 2.

Analysis of different diameter submucosal vessels. A, C, E, and G: percentage of vessels of different diameter in each patient with MNGIE and CTR. B, D, F, and H: representative images of sections stained with orcein for each vessel size are shown in (magnification bar 100 µm). Specifically, graph reporting the percentage of large diameter vessels in CTR vs. patients with MNGIE (*P = 0.0163) (A) with a representative image of large vessel (> 301 µm) in B. Graph illustrating the percentage of medium diameter vessels in CTR vs. patients with MNGIE (*P = 0.001) (C) with a representative image of medium vessel (300–101 µm) in D. Graph showing the percentage of small vessels (P = ns) (E) with a representative image of small vessel (100–51 µm) in F. Graph showing the percentage of the smallest diameter vessels in both groups (*P = 0.001) (G) with a representative image of the smallest diameter vessel (<50 µm) in H. MNGIE, mitochondrial neurogastrointestinal encephalomyopathy.

Figure 3.

Fibrosis assessment on full-thickness biopsies of jejunal tissue. A: the graph shows the fibrosis index in the full-thickness jejunal biopsies that was significantly increased in patients with MNGIE vs. controls (*P = 0.0043). Sections of the jejunum submucosa in CTR (B) and patients with MNGIE (C); note the abundant and disorganized spread of elastic fibers in patients with MNGIE illustrated in Ci that is a high magnification of C, which are absent in controls. Images were obtained from orcein-stained tissue sections (magnification bar 100 µm). Sections from CTR (D) and patients with MNGIE (E), that were stained using Sirius red and fast green. Red color is the collagen distributed in mucosa (M) and submucosa (SM), whereas light green represents noncollagenous fibers (magnification bar 100 µm). Sections from CTR (F) and patients with MNGIE (G) are stained with Sirius red and fast green to label vessel structure, particularly vessel wall (Wa) and endothelium (En) in green and labeled with *. Calibration bar: 50 µm. CTR, control; MNGIE, mitochondrial neurogastrointestinal encephalomyopathy.

Figure 4.

Longitudinal muscle quantitative and qualitative assessment. A: graph showing the difference in thickness of the external longitudinal muscle layer in controls (CTR) and patients with MNGIE (*P = 0.0033). Tissue sections from CTR (B) and patients with MNGIE (C), which are stained with Sirius red and fast green. The black double arrows in B and C indicate the thickness of the external longitudinal muscle layer (LML). Magnification bar: 500 µm. Section stained with Sirius red and fast green in CTR (D) and patients with MNGIE (E). In E, the majority of muscle cells (light green) of the LML are substituted by collagen fibers (red). The MP in patients with MNGIE is deeply enveloped in collagen. Note the increased collagen expression visible in the CML (magnification bar: 100 µm). CML, inner circular muscle layer; M, mucosa; MNGIE, mitochondrial neurogastrointestinal encephalomyopathy; MP, myenteric plexus; S, serosa; SM, submucosa.

Figure 5.

Myenteric plexus quantitative analysis. A: graph shows the number of neuronal cell bodies calculated per ganglion for each patient that was significantly decreased in patients with MNGIE vs. CTR (*P = 0.001). Neuron-specific enolase (NSE) immunoreactivity in tissue sections from CTR (B) and patients with MNGIE (C). The black arrows point to neuronal cell bodies (magnification bar: 50 µm). C from a biopsy of patients with MNGIE shows the entire ganglion, whereas B shows a portion of a ganglion from a control. B is a high magnification of the inset in the dotted area in Bi, which is a low-magnification image. In Bi, the two * indicate a distance lower than 300 µm, which is the cut-off we have previously established to define the arbitrary ganglionic unit in thin sections (17), thus NSE immunoreactive neurons within this area belong to the same arbitrary ganglionic unit (AGU) (17). D: graph shows the interganglionic distance calculated on neighboring ganglia for each patient, which is much higher in patients with MNGIE compared with CTR (*P = 0.001). The distance between adjacent ganglia visualized with NSE immunostaining in CTR (E) and patients with MNGIE (F). Black arrows indicate the distance between two ganglia in CTR (E) and patients with MNGIE (F). CTR, control; MNGIE, mitochondrial neurogastrointestinal encephalomyopathy.

Figure 6.

HIF-1α protein localization and expression. Tissue sections from CTR (A–C) and patients with MNGIE (D–F) stained with HIF-1α protein antibodies. Note the intense immunostaining in the longitudinal muscle layer (LML) (D), myenteric ganglia (E), and circular muscle layer (CML) (F) of patients with MNGIE compared with the lack of staining in CTR (A–C). Black arrows in E point to HIF-1α-positive neurons (magnification bar: 20 µm). G: the increase gradient of immunostaining intensity in the LML of tissue sections of patients with MNGIE from the circular muscle layer (CML, bottom) (lowest intensity) to the serosa (S, top) (highest intensity) (100 µm scale bar). H and I: the increase in HIF-1α jejunal protein expression in Western blotting from patients with MNGIE and CTR. HIF-1α jejunal protein expression was normalized vs. GAPDH (reference protein) and expressed in densitometry arbitrary unit (AU), *P < 0.001. CTR, control; HIF-1α, hypoxia-inducible-factor-1α; MNGIE, mitochondrial neurogastrointestinal encephalomyopathy.

Figure 7.

VEGF protein localization and expression. VEGF immunoreactivity in submucosal vessels in tissue sections from control (CTR) (A), and patients with MNGIE (B) (100 µm scale bar). High magnification images of submucosal vessels expressing VEGF immunoreactivity in tissue sections from CTR (C) and patients with MNGIE (D) (50 µm scale bar). Black arrows in D point to vessels with a fragmented wall. E: VEGF immunoreactive bands in Western blotting from CTR and patients with MNGIE jejunal tissues. F: quantification of VEGF jejunal protein expression normalized vs. VINCULIN (reference protein) and expressed in densitometry arbitrary unit (AU), *P < 0.05. MNGIE, mitochondrial neurogastrointestinal encephalomyopathy; VEGF, vascular endothelial cell growth factor.