Abstract

Osteoarthritis (OA) and obesity are prevalent health problems in today's society. Obesity increases the risk of OA in both weight-bearing joints, such as the knee, and non-weight-bearing joints, such as the hand. Additionally, obesity doubles the lifetime risk of developing OA. There is an urgent need for the identification of efficient therapeutic targets to address the issue of obesity-related osteoarthritis. In the context of obesity, adipose tissue secretes a large number of exosomes, which have been found to play a critical role in inter-organ communication and the regulation of joint functions. These exosomes contain micro-RNAs, small non-coding RNAs that act as guide molecules in RNA silencing. In our project, we utilize TargetScan software and a luciferase reporter plasmid to validate the target gene of obese exosomal micro-RNAs. By inhibiting the highly expressed adipocyte exosomal micro-RNAs, we aim to reduce their detrimental effects on the joints, making it a promising approach for anti-obesity and joint protection.

Obesity is a burgeoning global epidemic, with the World Obesity Federation estimating a rise from 2.6 billion affected individuals in 2020 to 4 billion by 2035 (Fig.1) ⁽¹⁾. Concurrently, osteoarthritis (OA) ranks as the most prevalent form of arthritis, particularly among older adults, and serves as one of the leading causes of physical disability in this age group ⁽²⁾. The condition is particularly widespread in China and the United States (Fig.2), which together account for a significant proportion of the 355 million global cases.

Fig.1 The predicted data of adult with obesity in the most populated countries from world obesity federation in 2035(Source: World Obesity Atlas 2023)

Fig.2 Arthritis will increase as the population grows and ages in USA(Source:National Health Interview Survey)

The intersection between obesity and OA is of critical interest. Obesity dramatically increases the risk of OA in both weight-bearing joints like the knees and in non-weight-bearing joints such as the hands ⁽³⁾. Obesity doubles the lifetime risk of symptomatic OA compared to individuals with a BMI below 25⁽⁴⁾. Coggon et al ⁽⁵⁾ reported that subjects with a BMI>30 kg/m2 were 6.8 times more likely to develop knee OA than normal-weight controls.

Inflammatory factors, revealed through elevated RNA expression levels of genes like TNF-α, IL-6, and IL-1β, have been found to be higher in the serum of obese individuals. This heightened inflammatory response contributes to the risk and potentially accelerates the progression of OA, especially in elderly patients.

Fig.3 Obesity affected OA in multiple ways (Source: fphys-09-00640-g004.jpg (frontiersin.org))

Adipose exosomes, secreted by adipose tissue, are pivotal players in cellular communication, affecting various physiological and pathological processes. They operate via autocrine, paracrine, and endocrine signaling pathways, and carry a variety of bioactive molecules like RNA, proteins, and lipids to target cells. Once delivered, these vesicles influence both gene transcription and protein modifications. Notably, white adipose tissue can induce insulin resistance and promote tumor growth through extracellular miRNAs ⁽⁶⁾. These vesicles also play a role in vascular remodeling as demonstrated by their impact on perivascular adipose tissue. Within these adipose exosomes, microRNAs (miRNAs) hold significant roles. These short, non-coding RNAs are involved in regulating gene translation and are implicated in multiple diseases, including cancer, diabetes, and obesity ⁽⁷⁾. In the context of obesity, exosomal miRNAs function as signaling agents, facilitating communication between adipose tissue and other organs such as the liver, skeletal muscles, and immune cells, leading to inflammation ⁽⁸⁾. Moreover, a decline in circulating miRNAs has been observed in lipodystrophy patients, indicating a strong association between adipose tissue and circulating miRNAs in humans ⁽⁹⁾.

Fig.4 Adipocyte-derived exosomes can enter the circulation and influence distant tissues (Source https://www.science.org/doi/10.1126/science.aaw6765)

In the team's previous research in 2022 (see detailed things in Human Practice) ⁽¹⁰⁾, our team student leader, Litong Wu examine the effect of obesity induced by high fat diet and adipose exosomes on knee post-traumatic osteoarthritis caused by DMM (destabilization of medial meniscus, DMM)surgery in young mice. Compared with the control diet group, high fat diet induced obesity can aggravate the pathological changes of the post-traumatic osteoarthritis caused by DMM surgery. Obese adipose exosomes can exacerbate the pathological changes of the knee articular cartilage.

More importantly, professor Zhao Yue from Nanjing University, our collaborator, utilized high-throughput miRNA sequencing to analyze obese adipocyte exosomes. The results highlighted 117 differentially expressed miRNAs across various Ad-Exos. Among them, seven exosomal miRNAs were found to be up-regulated in obesity but reduced after knockdown of the obesity-related genes in adipocytes. miR-3074-5p was of particular interest, exhibiting the most significant changes in expression levels.

Fig.5 High-throughput miRNA sequencing to predict the potential miRNAs that were altered in Ad-Exo (provided by Professor Zhaoyue)

Through literature reviews ^(11-15), as well as target gene prediction platforms, SMAD4 was confirmed as the regulatory target of miR-3074-5p. SMAD4, a critical factor in the TGF-β signaling pathway, is essential for bone and cartilage development. It has been reported that the expression of SMAD4 is downregulated in the articular cartilage of OA patients and that activating the TGF-β/SMAD4 pathway could mitigate the progression of diabetes-related OA ^(11-13). Moreover, overexpression of SMAD4 has been shown to inhibit LPS-induced chondrocyte apoptosis in vitro ⁽¹⁶⁾.

In our project, we have successfully followed the engineering design cycle to build new parts and identify potential targets for the treatment of osteoarthritis (OA) in obese subjects. We specifically focused on the mechanisms connecting adipose tissue to knee joint dysfunction, with a particular emphasis on the influence of adipose tissue-derived exosomes and their cargo microRNAs on OA in the context of obesity.

To identify potential targets, we employed TargetScan gene databases and pinpointed miR-3074-5p as a potential regulator of the SMAD4 gene. Subsequently, we designed a dual luciferase assay to investigate the binding impact of miR-3074-5p on the expression of SMAD4. For the experimental setup, we integrated the sequences of SMAD4 and the luciferase gene into the GV272 vector. Following transfection of the vector plasmid into E. coli DH5-alpha cells, the SMAD4 plasmids were fused with the luciferase gene. We then transfected chondrocytes with the luciferase-labeled SMAD4 plasmid (WT or Mut) and the miR-3074-5p plasmid (Mimics or NC) and assessed the fluorescence intensity of the chondrocytes to determine whether miR-3074-5p could bind SMAD4. Additionally, we investigated whether a specific inhibitor targeting miR-3074-5p in Ad-EVs (adipose tissue-derived extracellular vesicles) could alleviate pathological changes in chondrocytes by elucidating the regulatory relationship between miR-3074-5p and SMAD4.

In summary, our team, Nanjing-NFLS, is dedicated to confirming the significance of SMAD4 as a critical target that may be involved in miR-3074-5p-mediated OA in obese subjects. The goal of our project is to pave the way for innovative therapeutic strategies to anti-obesity and protect joint. Specifically, inhibiting miR-3074-5p could potentially alleviate the pathologic damage caused by exosomes from obese adipocytes, thereby offering a preventive approach to mitigate chondrocyte impairment in individuals with obesity-related OA.

Fig.6 Schematic experiment design in our project created with Biorender

Reference

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