Inhalation of hydrogen gas protects against mitomycin-induced pulmonary veno-occlusive disease.

BACKGROUND: As a subtype of pulmonary hypertension (PH), pulmonary veno-occlusive disease (PVOD) is devastating and life-threatening disease without effective therapy. Hydrogen has been reported to exhibits antioxidant and anti-inflammatory effects in a rat model induced by monocrotaline of PH. In this study, we investigated the effects of inhaled hydrogen gas on the prevention and treatment of PVOD induced by mitomycin C (MMC) in rats. METHODS: PVOD was induced in female Sprague-Dawley rats through intraperitoneal injection of MMC at a concentration of 3 mg·kg- 1·wk- 1 for 2 weeks. Inhalation of hydrogen gas (H2) was administered through a designed rat cage concurrently or two weeks after MMC administration. The severity of PVOD was assessed by using hemodynamic measurements and histological analysis. The expression levels of general control nonderepressible 2 (GCN2), nuclear factor erythroid 2-related factor-2 (Nrf2), heme oxygenase-1 (HO-1) and endothelial-to-mesenchymal transition (EndoMT) related proteins in lung tissue were measured. Levels of lipid peroxidation pro-inflammatory cytokines in serum were determined. RESULTS: Inhaled H2 improved hemodynamics and right heart function, reversed right ventricular hypertrophy, and prevented pulmonary vessel reconstitution in both prevention and treatment approaches. It decreased malondialdehyde (MDA) levels in the serum and the expression of NADPH oxidase 1 (NOX-1) in lung tissue. It regulated Nrf2/HO-1 signaling pathway and anti-inflammatory factor GCN2 in lung tissue, accompanied by a decrease in macrophages and pro-inflammatory cytokines. Our data suggested that H2 inhalation effectively countered EndoMT induced by MMC, as evidenced by the detection of endothelial markers (e.g., VE-cadherin and CD31) and mesenchymal markers (e.g., vimentin and fibronectin). Further research revealed that H2 preserved p-Smad3 and induced p-Smad1/5/9. CONCLUSION: Inhalation of H2 effectively inhibits the pathogenesis of PVOD induced by MMC in rats. This inhibitory effect may be attributed to the antioxidant and anti-inflammatory properties of H2.

The IL-33/ST2 Axis Promotes Primary Sjögren's Syndrome by Enhancing Salivary Epithelial Cell Activation and Type 1 Immune Response.

The molecular mechanisms of primary Sjögren's syndrome (pSS) are poorly understood. In this study, we explored the role of the IL-33/ST2 axis in the development of pSS. In the mouse model of experimental Sjögren's syndrome, we found that the saliva flow rate at weeks 4 and 30 was preserved in IL-33-/- and ST2-/- mice, compared with that of wild-type mice. At week 30 of experimental Sjögren's syndrome induction, the histological score, anti-nuclear Ab levels, and numbers of Th1 and B cells in draining lymph nodes of the salivary gland were lower in the IL-33-/- and ST2-/- mice, whereas Th17 cells and regulatory T cells were not changed. Primary salivary gland epithelial cells expressed the IL-33 receptor ST2. After stimulation with rIL-33, salivary gland epithelial cells increased the transcriptional levels of CD86 and CCL2, accompanied by the activation of the NF-κB inflammatory pathway. There was a synergistic effect between rIL-33 and rIL-12 in augmenting the production of IFN-γ in CD4+ T cells. In the pSS patients, the expression of IL-33 was elevated in the labial salivary gland, with the number of IL-33+ cells positively correlated with the score of the EULAR (European Alliance of Associations for Rheumatology) Sjögren's syndrome disease activity index (ESSDAI). ST2 was highly expressed in the cytoplasm of ductal epithelial cells, with low levels of expression in lymphatic infiltration sites. Our data suggest that the IL-33/ST2 axis may promote the development of pSS by enhancing salivary epithelial cell activation and the type 1 immune response.

[Pharmacological research progress of five classical prescriptions in treatment of chronic heart failure].

Chronic heart failure(CHF) is a comprehensive clinical syndrome caused by multiple factors that result in structural and/or functional abnormalities of the heart, leading to impaired ventricular contraction and/or relaxation functions. This medical condition represents the final stage of various cardiovascular diseases. In the treatment of CHF, multiple clinical studies have demonstrated the benefits of using traditional Chinese medicine(TCM) to control oxidative stress, inflammation, and apoptosis, thereby delaying ventricular remodeling and reducing myocardial fibrosis. In this study, common TCM syndromes in the diagnosis and treatment of CHF in recent years were reviewed and summarized. Five common treatment methods including benefiting Qi and activating blood circulation, enhancing Qi and nourishing Yin, warming Yang for diuresis, eliminating phlegm and dampness, rescuing from collapse by restoring Yang, and corresponding classic prescriptions in prevention and treatment of CHF were concluded under the guidance of TCM syndrome differentiation thinking. Meanwhile, research progress on the modern pharmacological effects of these classic prescriptions was systematically discussed, so as to establish a unique treatment system for CHF by classic prescriptions under the guidance of TCM syndrome differentiation theory and provide innovative diagnosis and treatment strategies for clinical CHF.

[Advances in animal models of chronic heart failure and its applications in traditional Chinese medicine].

Chronic heart failure(CHF) is a series of clinical syndromes in which various heart diseases progress to their end stage. Its morbidity and mortality are increasing year by year, which seriously threatens people's life and health. The diseases causing CHF are complex and varied, such as coronary heart disease, hypertension, diabetes, cardiomyopathy and so on. It is of great significance to establish animal models of CHF according to different etiologies to explore the pathogenesis of CHF and develop drugs to prevent and treat CHF induced by different diseases. Therefore, based on the classification of the etiology of CHF, this paper summarizes the animal models of CHF widely used in recent 10 years, and the application of these animal models in traditional Chinese medicine(TCM) research, in order to provide ideas and strategies for studying the pathogenesis and treatment of CHF, and provide ideas for TCM modernization research.

[Progress of target determination and mechanism of bioactive components of traditional Chinese medicine].

The pharmacodynamic substances of traditional Chinese medicine(TCM) are the basis for the research of TCM and the development of innovative drugs. However, the lack of clarity of targets and molecular mechanisms is the bottleneck problem that restricts the research of pharmacodynamic substances of TCM. Bioactive components are the material basis of the efficacy of TCM, which exert activity by regulating the corresponding targets. Therefore, it is very important to identify the targets of the bioactive components to elucidate the pharmacological mechanism of TCM. Proteins are the most important drug targets, and study of the interaction between the proteins and bioactive components of TCM plays a key role in the development of pharmacological mechanism of TCM. In recent years, the main techniques for detecting the interaction between the bioactive components and proteins include surface plasmon resonance, fluorescence resonance energy transfer, bio-layer interference, molecular docking, proteome chip, target fishing, target mutant, and protein crystallization techniques, etc. This review summarized the biological target detection techniques and their applications in locating the targets of the bioactive components in TCM in the last decade, and this paper will provide useful strategies to elucidate the pharmacological mechanisms of TCM.

Clinical features of rheumatic patients infected with COVID-19 in Wuhan, China.

OBJECTIVE: The clinical features of rheumatic patients with coronavirus disease 2019 (COVID-19) have not been reported. This study aimed to describe the clinical features of COVID-19 in rheumatic patients and provide information for handling this situation in clinical practice. METHODS: This is a retrospective case series study. Deidentified data, including gender, age, laboratory and radiological results, symptoms, signs, and medication history, were collected from 2326 patients diagnosed with COVID-19, including 21 cases in combination with rheumatic disease, in Tongji Hospital between 13 January and 15 March 2020. RESULTS: Length of hospital stay and mortality rate were similar between rheumatic and non-rheumatic groups, while the presence of respiratory failure was more common in rheumatic cases (38% vs 10%, p<0.001). Symptoms of fever, fatigue and diarrhoea were seen in 76%, 43% and 23% of patients, respectively. There were four rheumatic patients who experienced a flare of rheumatic disease during hospital stay, with symptoms of muscle aches, back pain, joint pain or rash. While lymphocytopaenia was seen in 57% of rheumatic patients, only one patient (5%) presented with leucopenia in rheumatic cases. Rheumatic patients presented with similar radiological features of ground-glass opacity and consolidation. Patients with pre-existing interstitial lung disease showed massive fibrous stripes and crazy-paving signs at an early stage. Five rheumatic cases used hydroxychloroquine before the diagnosis of COVID-19 and none progressed to critically ill stage. CONCLUSIONS: Respiratory failure was more common in rheumatic patients infected with COVID-19. Differential diagnosis between COVID-19 and a flare of rheumatic disease should be considered. TRIAL REGISTRATION NUMBER: ChiCTR2000030795.

Prior cervical cytology and high-risk HPV testing results for 311 patients with invasive cervical adenocarcinoma: a multicenter retrospective study from China's largest independent operator of pathology laboratories.

BACKGROUND: High-risk human papillomavirus (HR-HPV) testing is more sensitive than cytology for the detection of cervical cancer and its precursors. However, limited and inconsistent data are available about the efficacy of the combination of these two methods for screening cervical adenocarcinoma. This multicenter retrospective study investigated the screening results of a cohort of Chinese patients who were subsequently diagnosed with invasive cervical adenocarcinoma, with the goal of identifying the optimal cervical adenocarcinoma screening method. METHODS: We retrospectively retrieved and analyzed the data from patients with histologically confirmed primary invasive cervical adenocarcinoma from eight local pathology laboratories operated by KingMed Diagnostics, the largest independent operator of pathology laboratories in China, over a 2-year period. Only patients who underwent cytology and/or HR-HPV testing within 6 months before the adenocarcinoma diagnosis were included. HR-HPV DNA was detected using one of two HPV test kits: the Hybrid Capture 2 (HC2) assay (Qiagen, Hilden, Germany) and an HPV genotyping panel (Yaneng Bio, Shenzhen, China). RESULTS: Of the 311 patients, 136 underwent cytology alone, 106 underwent HR-HPV testing alone, and 69 underwent cytology and HR-HPV co-testing. The sensitivities of cytology alone (64.0, 95% confidence interval [CI]: 55.9-72.0) and HR-HPV testing alone (66.0, 95% CI: 57.0-75.1) were similar (P = 0.738). The sensitivity of cytology and HR-HPV co-testing (87.0, 95% CI: 79.0-94.9) was significantly higher than that of either cytology (P = 0.001) or HR-HPV testing alone (P = 0.002). CONCLUSIONS: Both cytology alone and HR-HPV testing alone showed poor screening efficiency, whereas the combination of the two clearly increased the efficiency of primary cervical adenocarcinoma screening. Thus, cytology and HR-HPV co-testing might be the most efficient cervical adenocarcinoma screening method.

[Value of early application of different doses of amino acids in parenteral nutrition among preterm infants].

OBJECTIVE: To study the short-term response and tolerance of different doses of amino acids in parenteral nutrition among preterm infants. METHODS: This study included 86 preterm infants who had a birth weight between 1 000 to 2 000 g and were admitted to the hospital within 24 hours of birth between March 2013 and June 2014. According to the early application of different doses of amino acids, they were randomized into low-dose group (n=29, 1.0 g/kg per day with an increase of 1.0 g/kg daily and a maximum of 3.5 g/kg per day), medium-dose group (n=28, 2.0 g/kg per day with an increase of 1.0 g/kg daily and a maximum of 3.7 g/kg per day), and high-dose group (n=29, 3.0 g/kg per day with an increase of 0.5-1.0 g/kg daily and a maximum of 4.0 g/kg per day). Other routine parenteral nutrition and enteral nutrition support were also applied. RESULTS: The maximum weight loss was lower and the growth rate of head circumference was greater in the high-dose group than in the low-dose group (P<0.05). The infants in the medium- and high-dose groups had faster recovery of birth weight, earlier attainment of 100 kcal/(kg·d) of enteral nutrition, shorter duration of hospital stay, and less hospital cost than those in the low-dose group (P<0.05). Blood urea nitrogen (BUN) levels in the high-dose group increased compared with the other two groups 7 days after birth (P<0.05). The levels of creatinine, pH, bicarbonate, bilirubin, and transaminase and the incidence of complications showed no significant differences between groups (P>0.05). CONCLUSIONS: Parenteral administration of high-dose amino acids in preterm infants within 24 hours after birth can improve the short-term nutritional status of preterm infants, but there is a transient increase in BUN level.

Disorder of neuroplasticity aggravates cognitive impairment via neuroinflammation associated with intestinal flora dysbiosis in chronic heart failure.

BACKGROUND: Chronic heart failure (CHF) impairs cognitive function, yet its effects on brain structure and underlying mechanisms remain elusive. This study aims to explore the mechanisms behind cognitive impairment. METHODS: CHF models in rats were induced by ligation of the left anterior descending coronary artery. Cardiac function was analyzed by cardiac ultrasound and hemodynamics. ELISA, immunofluorescence, Western blot, Golgi staining and transmission electron microscopy were performed on hippocampal tissues. The alterations of intestinal flora under the morbid state were investigated via 16S rRNA sequencing. The connection between neuroinflammation and synapses is confirmed by a co-culture system of BV2 microglia and HT22 cells in vitro. Results: CHF rats exhibited deteriorated cognitive behaviors. CHF induced neuronal structural disruption, loss of Nissl bodies, and synaptic damage, exhibiting alterations in multiple parameters. CHF rats showed increased hippocampal levels of inflammatory cytokines and activated microglia and astrocytes. Furthermore, the study highlights dysregulated PDE4-dependent cAMP signaling and intestinal flora dysbiosis, closely associated with neuroinflammation, and altered synaptic proteins. In vitro, microglial neuroinflammation impaired synaptic plasticity via PDE4-dependent cAMP signaling. CONCLUSIONS: Neuroinflammation worsens CHF-related cognitive impairment through neuroplasticity disorder, tied to intestinal flora dysbiosis. PDE4 emerges as a potential therapeutic target. These findings provide insightful perspectives on the heart-gut-brain axis.

Airway delivery of Streptococcus salivarius is sufficient to induce experimental pulmonary hypertension in rats.

BACKGROUND AND PURPOSE: The causal relationship between altered host microbiome composition, especially the respiratory tract microbiome, and the occurrence of pulmonary hypertension (PH) has not yet been studied. An increased abundance of airway streptococci is seen in patients with PH compared with healthy individuals. This study aimed to determine the causal link between elevated airway exposure to Streptococcus and PH. EXPERIMENTAL APPROACH: The dose-, time- and bacterium-specific effects of Streptococcus salivarius (S. salivarius), a selective streptococci, on PH pathogenesis were investigated in a rat model established by intratracheal instillation. KEY RESULTS: Exposure to S. salivarius successfully induced typical PH characteristics, such as elevated right ventricular systolic pressure (RVSP), right ventricular hypertrophy (Fulton's index) and pulmonary vascular remodelling, in a dose- and time-dependent manner. Moreover, the S. salivarius-induced characteristics were absent in either the inactivated S. salivarius (inactivated bacteria control) treatment group or the Bacillus subtilis (active bacteria control) treatment group. Notably, S. salivarius-induced PH is characterized by elevated inflammatory infiltration in the lungs, in a pattern different from the classic hypoxia-induced PH model. Moreover, in comparison with the SU5416/hypoxia-induced PH model (SuHx-PH), S. salivarius-induced PH causes similar histological changes (pulmonary vascular remodelling) but less severe haemodynamic changes (RVSP, Fulton's index). S. salivarius-induced PH is also associated with altered gut microbiome composition, suggesting potential communication of the lung-gut axis. CONCLUSION AND IMPLICATIONS: This study provides the first evidence that the delivery of S. salivarius in the respiratory tract could cause experimental PH in rats.

3-Meth-oxy-4-methyl-1H-1,2,4-triazol-5(4H)-one monohydrate.

In the title hydrate, C(4)H(7)N(3)O(2)·H(2)O, all the non-H atoms lie on a crystallographic mirror plane. The H atoms of both methyl groups are disordered over two sets of sites. In the crystal, N-H⋯O(w) and O(w)-H⋯O(k) (w = water and k = ketone) hydrogen bonds link the components into (010) sheets.

4-[(2-Bromo-benzyl-idene)amino]-3-(pyridin-4-yl)-1H-1,2,4-triazole-5(4H)-thione.

In the title compound, C(14)H(10)BrN(5)S, the dihedral angle between the triazole ring and the pyridine and bromo-benzene rings are 26.42 (13) and 6.28 (13)°, respectively. The molecule exists as a thione in the solid state. In the crystal, mol-ecules are linked by N-H⋯N hydrogen bonds, generating [010] C(8) chains.

2,2'-(Piperazine-1,4-di-yl)diacetonitrile.

The complete mol-ecule of the title compound, C(8)H(12)N(4), is generated by a crystallographic inversion centre. The piperazine ring adopts a chair conformation with the N-bonded substituents in equatorial positions. In the crystal, mol-ecules are linked by C-H⋯N(c) (c = cyanide) hydrogen bonds.

IL-33/ST2 axis contributes to the dermal fibrosis of systemic sclerosis via promoting fibroblasts activation.

BACKGROUND: Systemic sclerosis (SSc) is a chronic immune-mediated rheumatic disease that is characterized by fibrosis of the skin and internal organs. Interleukin-33 (IL-33) has been recently implicated in several autoimmune diseases through its receptor ST2. OBJECTIVE: The aim of this study was to investigate the role and underlying mechanism of IL-33/ST2 axis in the fibrotic disorder of SSc. METHODS: The bleomycin (BLM)-induced fibrotic skin and skin biopsies of SSc patients were used to detect the expression of IL-33 and ST2. Human dermal fibroblasts were stimulated with recombinant IL-33(rIL-33) protein and their activation, proliferation and migration were assessed. The role of IL-33/ST2 axis was investigated in mouse fibrosis model via histologically assessing skin fibrosis after IL-33 gene knockout. ST2 neutralizing antibody treatment was also obtained to estimate the possible effect. RESULTS: The number IL-33+ cells and ST2+ cells were increased in the lesion skin of SSc patients and BLM-induced mouse. Human skin fibroblasts highly expressed ST2 protein, and the proliferation, migration, and collagen expression were significantly elevated after rIL-33 stimulation, accompanied by the activation of MAPKs and NF-kB pathways. The severity of skin fibrosis was significantly reduced in il33-/- mice compared with WT mice. Blockade of IL-33 receptor using an anti-ST2 neutralizing antibody effectively ameliorated the skin fibrosis. CONCLUSION: These data indicate that IL-33/ST2 axis contributes to the fibrotic skin injury of SSc via promoting fibroblasts activation, and IL-33/ST2 blockade might serve as a novel strategy to inhibit the fibrosis progression in patients of SSc.

2-Meth-oxy-N-[(S)-3-methyl-butan-2-yl]-6-{[(S)-3-methyl-butan-2-yl]amino}-3,5-dinitro-benzamide.

The title compound, C(18)H(28)N(4)O(6), crystallizes with two mol-ecules in the asymmetric unit which differ slightly in conformation. The dihedral angle between the amide plane and the benzene ring are 72.6 (2) and 66.8 (2)° in the two mol-ecules. A strong intra-molecular N-H⋯O hydrogen bond between the amino and nitro groups occurs in each mol-ecule. The crystal structure features two symmetry-independent polymeric chains along [010] generated by N-H⋯O hydrogen bonds between the amide groups.

Methyl 2-(4-chloro-3,5-dinitro-benz-amido)-acetate.

The title mol-ecule, C(10)H(8)ClN(3)O(7), is twisted with the dihedral angle between the amide and benzene ring being 38.75 (11)°. The C-N-C-C torsion angle between the amide and acetyl groups is -150.1 (2)°. Finally, each nitro group is twisted out of the plane of the benzene ring to which it is connected [O-N-C-C torsion angles = 34.0 (3) and -64.5 (3)°]. Linear supra-molecular chains along [010] and mediated by N-H⋯O hydrogen bonds between successive amide groups dominate the crystal packing. The chains are consolidated into the three-dimensional structure by C-H⋯O contacts.

[Preparation and anti-oxidant activity of cinnamic acid derivatives-g-CTS].

OBJECTIVE: To prepare cinnamic acid derivatives-g-CTS and to study its antioxidation activity. METHOD: The ability of catching oxygen of the products and raw material were determined through two methods, Marklund method and trace pyrogallic acid method, with autoxidation reaction of pyrogallol as the oxygen anion source. RESULT: The antioxidation activities of all products were better than the raw material. CONCLUSION: Cinnamic acid derivatives-g-CTS is suitable as the O2-* -capture agent.

Gut Microbiota Aberration in Patients of Systemic Sclerosis and Bleomycin-Induced Mice Model.

Systemic sclerosis (SSc) is an immune-mediated systemic autoimmune disease with unknown etiology, which has high morbidity and mortality. Current treatments to dispose of this disorder are limited. And there are still no ideal animal models that can fully replicate the four basic pathophysiological features of SSc, including vascular lesions, fibrosis, inflammation, and autoimmunity, let alone animal models specifically designed to study gastrointestinal lesions. It's essential to seek and establish appropriate animal models to explore the role of gut microbiota in the pathogenesis of SSc. In this study, we found similar gut microbiota aberration in patients of SSc and bleomycin (BLM)-induced mice model through 16S rRNA gene sequencing. In terms of phylum-level differences, the relative abundance of Bacteroidetes was significantly decreased and Firmicutes increased in the SSc patients and the mice. Notably, the genera of Lactobacillus, commonly used as a probiotic additive, was also elevated in SSc patients and BLM mice, which was consistent with a few of studies. Therefore, the model can likely mimic the pathological changes of gut microbiota in patients with SSc, which may offer an important potential platform for the in-depth understanding of gut microbiota aberration in patients with SSc and to devise potential disease-modifying treatments.

Intratracheal Poly(I:C) Exposure Accelerates the Immunological Disorder of Salivary Glands in Sjogren's-Like NOD/ShiLtJ Mice.

Evidences have suggested that Sjogren's syndrome (SS) is associated with viral infection. The aim of this study was to investigate the involvement of respiratory viral poly(I:C) in the pathogenesis of SS and potential mechanisms using a SS-like NOD/ShiLtJ (NOD) mouse model. 5-week female NOD mice were intratracheally administered poly(I:C) every other day for 5 times to mimic viral infection. Pilocarpine induced saliva secretion was determined every 8 days. Submandibular glands (SMG) and lungs were harvested for the detection of pathological changes. We found that intratracheal administration of poly(I:C) significantly advanced and enhanced the reduction of saliva flow rate in NOD mice. Furthermore, poly(I:C) treatment aggravated the histopathological lesions and inflammatory cells infiltration in SMG. Accompanied by elevated expression of IFN cytokines and IL-33, Th1 activation was enhanced in SMG of poly(I:C)-treated NOD mice, but Th17 cells activation was unchanged among the groups. In addition, intratracheal poly(I:C) exposure promoted the expression of IL-33 and increased T cells proportion in the lung, which were consistent with the change in SMG. Therefore, intratracheal poly(I:C) exposure aggravated the immunological and function disorder of SMG in NOD mice.

Mitomycin C induces pulmonary vascular endothelial-to-mesenchymal transition and pulmonary veno-occlusive disease via Smad3-dependent pathway in rats.

BACKGROUND AND PURPOSE: Pulmonary veno-occlusive disease (PVOD) is a rare disease characterized by the obstruction of small pulmonary veins leading to pulmonary hypertension. However, the mechanisms underlying pulmonary vessel occlusion remain largely unclear. EXPERIMENTAL APPROACH: A mitomycin C (MMC)-induced PVOD rat model was used as in vivo animal model, and primarily cultured rat pulmonary microvascular endothelial cells (PMVECs) were used as in vitro cell model. KEY RESULTS: Our data suggested an endothelial-to-mesenchymal transition (EndoMT) may be present in the pulmonary microvessels isolated from either PVOD patients or MMC-induced PVOD rats. In comparison to the control vessels, vessels from both PVOD patients and PVOD rats had co-localized staining of specific endothelial marker von Willebrand factor (vWF) and mesenchymal marker α-smooth muscle actin (α-SMA), suggesting the presence of cells that co-express endothelial and mesenchymal markers. In both the lung tissues of MMC-induced PVOD rats and MMC-treated rat PMVECs there were decreased levels of endothelial markers (e.g. VE-cadherin and CD31) and increased mesenchymal markers (e.g. vimentin, fibronectin and α-SMA) were detected indicating EndoMT. Moreover, MMC-induced activation of the TGFß/Smad3/Snail axis, while blocking this pathway with either selective Smad3 inhibitor (SIS3) or small interfering RNA (siRNA) against Smad3, dramatically abolished the MMC-induced EndoMT. Notably, treatment with SIS3 remarkably prevented the pathogenesis of MMC-induced PVOD in rats. CONCLUSIONS AND IMPLICATIONS: Our data indicated that targeted inhibition of Smad3 leads to a potential, novel strategy for PVOD therapy, likely by inhibiting the EndoMT in pulmonary microvasculature.