Echography analysis of musculoskeletal, heart and liver alterations associated with endothelial dysfunction in obese rats

Overweight and obesity are defined as an abnormal or excessive accumulation of fat that is harmful to health, and are an important risk factor for non-communicable diseases, such as cardiovascular diseases (mainly heart disease and strokes); moreover, overweight/obesity is associated with metabolic disorders (glucose intolerance, insulin resistance, hyperlipidemia, diabetes and hypertension), musculoskeletal disorders (especially osteoarthritis) and some cancers (endometrial, breast, ovarian, prostate, liver, gallbladder, kidney and colon) [1].

Some mechanisms that are triggered by obesity involve the inflammatory process, which includes the production of proinflammatory cytokines such as interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α). Thus, the transendothelial migration of inflammatory cells (leucocytes) and the production of cytokines are an early step in endothelial dysfunction, which continues with the activation of adhesion molecules (ICAM-1, VCAM-1); this contributes to the progression from early endothelial dysfunction to atherosclerotic plaques, causing vascular complications [2‐6]. To understand these pathophysiological mechanisms, experimental models are used, and these approaches include murine models (rats). Which is valuable tool for uncovering the mechanisms linked to the comorbidities of the metabolic disease [7].

In obese rats, excess lipid accumulates in other tissues, including the liver, skeletal muscle and heart, and this condition is associated with an increase in adipose mass and free fatty acids. Likewise, in the liver, this accumulation along with other intrahepatic signals leads to a derangement in glucostatic and lipidostatic functions, and generate a greater vulnerability to hepatic steatosis [8, 9].

Echography is commonly used in the clinical setting for the diagnosis and follow-up of patients with nonalcoholic fatty liver disease (NAFLD); in addition, this analysis is a good method that allows the examination of arteries, cardiopathies and fatty tissue [10]. However, no study has used murine models to assess the sonographic findings of several organs in obesity, which could be identified as the integration and relationship of dysfunctions in various mechanisms in other organs or comorbidities with in the same organism.

Ultrasound is an economical, accessible, fast, precise, simple, comfortable, and noninvasive procedure that does not cause pain or involve, radiation. In addition, it obtains images with high sensitivity and accuracy and is essential in the study of a variety of organs, such as the liver and the musculoskeletal system. Two-dimensional echocardiography is the study of the heart in two dimensions; it allows us to analyze the organ as a whole and the relationships that the cardiac structures maintain with each other. Two-dimensional echocardiography is very useful for the study of congenital anomalies, the differentiation between thrombi and intracardiac masses, and the analysis of regions that are difficult to access via the one-dimensional echocardiogram [11].

Although there are ultrasound studies in rats where the various morphological and functional aspects are analyzed at the cardiac level, mainly in the left ventricle (LV), none of these studies has been considered important for a joint analysis of both, the LV and the hepatic, renal and musculoskeletal systems represented in humans [12]. Therefore, we herein report the echographic analysis of the left ventricle, hepatic, musculoskeletal disorders and endothelial dysfunction in obesity.

Figure 1 shows the differences in food consumption in the study groups: the obese group fed a hypercaloric diet exhibited an increased food intake compared with that of the control group (p < 0.001). Additionally, an increase in body weight was observed in the Ob group vs the C group (22.5%; 472 ± 25 vs 385 ± 20, p < 0.001) and the Ob group showed an increase in fasting glucose level (p < 0.05), lipid profile and HOMA-IR index (300%; 4 ± 1 vs 16 ± 3, p < 0.0001). Therefore, because of the lipid analysis, these rats were considered obese rats with metabolic alterations (Table 1).

This experimental model demonstrated endothelial dysfunction, manifested by the inflammatory cytokine TNF-α as well as by adhesion molecules that are indicators of endothelial damage, such as ICAM-1 (87.6%, 122 ± 9 vs 65 ± 5, p < 0.0001) and VCAM-1 (29.4% 110 ± 10 vs 85 ± 2, p < 0.05) (Table 2, Fig. 2c, d). ICAM-1 and VCAM-1 are relevant to chronic inflammatory processes, with an increased risk for type 2 diabetes (T2DM) and cardiovascular diseases (CVD) [6, 7, 14].

In the Ob group, an increase in these adhesion molecules and in proinflammatory cytokines such as TNF-α was demonstrated, with a significant difference compared with the C group (220%, 16 ± 2 vs 5 ± 0.05, p < 0.001); a similar increase was observed for IL-6 (155%, 23 ± 5 vs 9 ± 1, p < 0.001) (Table 2). According to the measurements of the metabolic parameters and cytokines, the Ob group, which consumed hypercaloric diet, presented endothelial damage. In addition, modern imaging provided an integral approach to evaluate the control group (Table 3; Fig. 2a, b), in which the aorta and LV were observed. For LV, an increase in the septum and wall was found in the Ob group (Table 3).

However, in Fig. 3, the analysis of the two-dimensional ultrasound was more complete, revealing fat infiltration with the presence of a fatty liver (Table 3; Fig. 3b) in the obese group; on the other hand, an isoechoic homogeneous hepatic parenchyma was found in the control group (Fig. 3a).

In another analysis of the ultrasound dimensions, the Ob group showed renal pelvic dilatation compared to the control group, in which the kidney was echogenic, with no evidence of dilation (Fig. 3c, d).

In the Ob group, the ultrasound images revealed musculoskeletal alterations that were due to fat deposits and demonstrated the presence of articular cysts, in the posterior region of the distal joints, where it was possible to view the damage caused the accumulated fat (Table 3; Fig. 3e, f).

In this study, we show the relationships among obesity, endothelial dysfunction and alterations in several organs, such as the musculoskeletal system, kidney, liver and heart, through noninvasive methods such as echography. Thus, our results revealed that a high fat-fructose diet increased food intake, with marked weight gain due to the caloric contribution provided by fat and carbohydrates in obese group. We also found metabolic alterations, such as increases in glucose, triglycerides, cholesterol and the HOMA-IR index, similar to other studies that have used hypercaloric diets [13, 15]. Hence, our results showed that with obesity triggered dysmetabolism, hyperglycemia and hypertriglyceridemia, as well as an increase of the levels of inflammatory cytokines (TNF-α, IL-6), are present. Likewise, as a consequence of obesity, alterations in other organs can manifest and these changes can be analyzed by ultrasound. In this work the results at the muscle-skeletal and articular levels showed the presence of articular cysts, in the posterior region of the distal joints, where it was possible to view the damage due to fat accumulation. Other alterations were present in the heart, kidney, musculoskeletal system and liver analyzed by echography.

In this study, we used a noninvasive and rapid method, to evaluate the ventricular anatomy. Although the cut-off point for normal relative parietal thickness in humans is 0.42 mm, this criterion was highly specific in our animals because the rats presented a relative parietal thickness below that limit [11, 12]. Additionally, in this work the presence of Baker’s cyst (QB), or popliteal cyst, was first described in 1840 by Adams [16] and later by Baker [17]. In 1877, Baker published his experience with this entity, which caused this type of cyst to be named after him. Baker’s cyst is defined as an abnormal cluster of synovial fluid in the gastrocnemius-semimembranosus bursa or as a herniation of the posterior joint capsule with synovial fluid tension [17‐19]. Changes in both static and dynamic alignment of the lower extremities could alter balance and gait, and trigger pain throughout the lower limbs [20]. In addition, ultrasound changes in the liver indicated fatty liver in 75% (6 exemplary) of the obese rats. One of the main organs affected by obesity is the liver, where long-term increases in lipogenesis and decreases in mitochondrial β-oxidation of nonesterified fatty acids, as well as hepatic triglyceride secretion, can contribute to fat accumulation in the liver, leading to the appearance of liver steatosis. Additionally, in a prospective longitudinal study, 86% of patients with NAFLD and progressive fibrosis were obese [14, 15, 21].

At the renal level, changes such as pelvic dilation were found in 50% of the obese group rats. On the other hand, changes were observed at the level of the musculoskeletal system with the presence of joint cysts in the posterior region of the distal lower-extremity joints of the rodents. Thus, excessive body weight creates greater load stress, which causing joint misalignment (deformities) in the lower extremities and inflammatory and degenerative processes. This condition could decrease physical functioning due to associations with mobility and pain [22].

Several studies have shown that the mechanical stress caused by overload or repetitive use can trigger tendon pathology. Moreover, certain extrinsic factors (posture and activity) and intrinsic factors (genetics and metabolic characteristics) can interfere with their development [16]. This condition and metabolic factors (hyperglycemia, dyslipidemia and endothelial dysfunction) affect the quality of life of subjects.

Regarding the inflammatory process and endothelial dysfunction, we also found an increase in proinflammatory cytokines, such as TNF-α and IL-6, which augment monocytes adhesion to endothelial cells. It is known that in both; humans and mice, an imbalance between TNF-α and adiponectin (an adipokine of adipose tissue) participates in the progression to steatohepatitis [23‐25]. Therefore, adipose tissue releases inflammatory mediators, proinflammatory molecules such as TNF-α and IL-6, and some other mediators such leptin, adiponectin, and resistin. IL-6, TNF-α and leptin act on immune cells and cause local and systemic inflammation. In several studies, endothelial damage has been demonstrated to augment molecules such as monocyte attractant chemoprotein 1 (MCP-1), ICAM-1, VCAM-1 and plasminogen activator inhibitor (PAI-1), which contribute to vascular complications [6, 7, 26, 27].

Thus, our results of adhesion molecules showed an increase in the Ob group; this confirms endothelial damage, which has an impact on the joints. In addition, ICAM-1 and VCAM-1 are known to be activating molecules of endothelial dysfunction because they play a crucial role in the adhesion of cells to the endothelial surfaces and in the integrity of the vascular wall, generating an accumulation of cells and sparking oxidative stress, which can be modulated by the body composition and eating pattern [4, 6, 28, 29].

In this study, we observed a relationship between endothelial dysfunction and the changes observed at the level of the musculoskeletal system, liver and heart with the presence of articular cysts in the posterior region of the distal lower-extremity joint in obese rodents. Thus, we suggest that ultrasound is an excellent diagnostic tool that is accessible and easy to use to access the chronic diseases that are increasing in our population. Moreover, ultrasound allows an integral assessment of the alterations in different organs, joints and tissues, as well as, better monitoring and support to reduce complications and improve the quality of life of patients.