II. Mouse model OA, systemic rapamycin

Autophagy Activated by Rapamycin Reduces severity of Experimental OA

Carames, Lotz, 2012, Scripps Institute La Jolla, Ca, Spain.


Abstract

Methods: Experimental OA was induced by transection of medial knee ligament in 2 month old mouse. Rapamycin was administered for 10 weeks with injection 1 mg/kg weight/day.

Histopathological changes in articular cartilage and synovium were examined. Rapamycin effects on mTOR signaling, autophagy, cartilage homeostasis and inflammation were analyzed.


Objectives: Osteoarthritis (OA) is associated with cell death and extracellular matrix degradation in articular cartilage. Autophagy is an essential mechanism that was found to be deficient in aging and OA cartilage. This study determined whether pharmacological inhibition of mTOR, a key inhibitor of autophagy, has disease-modifying activity in experimental OA.


Results: Rapamycin lowered mTOR signaling pathway in mouse knee joints...and caused activation of LC3, a main marker of autophagy. The severity of cartilage degradation was significantly reduced in the rapamycin treated group..this was associated with a decrease in synovitis. Rapamycin treatment also maintained cartilage cellularity and decreased ADAMTS-5 and IL-1B expression in articular cartilage.


[NOTE: ADAMTS-5 is key enzyme which dissolves cartilage resulting in proteolytic matrix destruction.


IL-1B is key inflammatory substance.]


Conclusions: "These results suggest that rapamycin, at least in part by autophagy activation, reduces the severity of experimental OA. Pharmacological activation of autophagy may be an effective therapeutic approach for OA."


[Note: the dose used, 1 mg/kg/day by injection is equivalent to oral dose  of 4 mg/kg a day in mice.


Intermittent dose of 3-6 mg once a week in humans should achieve comparable blood levels of rapamycin.]



 

Introduction

"Osteoarthritis (OA), the most common aging-related joint pathology, is characterized by degradation of cartilage extracellular matrix (ECM) and reduced cartilage cellularity."


mTOR is an important suppressor of autophagy. 


"In articular cartilage, which is characterized by a very low rate of cell turnover, autophagy would appear to be essential to maintain cell survival and function...Reduced expression of autophagy regulators was observed in joint aging and OA in humans and mice, and this was accompanied by an increase in chondrocyte apoptosis" [chondrocyte death].


"Collectively, these observations suggest that compromised autophagy may contribute to the development of OA. The objective of this study was to establish proof-of-principle that pharmacological enhancement of autophagy may have a disease-modifying activity in experimental OA."

Results

1. Systemic rapamycin decreased mTOR signaling in mouse knee joints in articular cartilage and menisci.

Rapamycin increased LC3 expression, showing strong activaton of autophagy in articular cartilage.


2. Rapamycin reduces severity of experimental OA.

Investigators performed surgery on mouse knee joint by transcection of medial ligaments. [An experimental  method to produce OA in mouse knee joint.] They gave injections of rapamycin 1mg/kg body weight daily for 10 weeks and then evaluated. Rapamycin treated mice had significant decrease in carlilage pathology by approximately 50%.


3. Rapamycin maintains cartilage cellularity and reduces ADAMTS-5 expression in experimental OA.

This was to determine mechanism of action. ADAMTS-5 is main proteinase for aggrecan degradation in articular cartilage. 


Rapamycin protected against loss of chondrocytes, the cartilage cell.

"These finding show that rapamycin protects against cell loss and extracellular matrix damage by reducing ADAMTS-5 expression."


4. Rapamycin reduced Inflammation

Histopathological exam of synovial tissue showed reduced inflammation. 

Rapamycin reduced synovial level of IL-1B.





Discussion

The study was to determine whether activation of autophagy can reduce severity of experimental OA. Rapamycin reduces signaling of mTOR and increases autophagy. 


Our results show that systemic administration of rapamycin inhibited mTOR signaling. Rapamycin activated autophagy as shown by increase in LC3-II expression. 


In experimental OA model, rapamycin caused a significant reduction in OA severity by approximately 50%. To address potential mechanism, we examined cartilage cellularity. The number of chondrocytes was increased compared to controls.


ADAMTS-5, a major aggrecan-degrading exzyme had decreased expression in  rapamycin treated mice.


OA in humans is associated with inflammatory changes in synovium. In present sudy, rapamycin reduced severity of synovitis. This was associated with reduction of IL-1B.


The present study is the first to establish efficacy of rapamycin in an animal model of OA.


"In summary, we report that rapamycin, at least in part by autophagy activation, reduces severity of experimental OA. These results suggest that pharmacological inhibition of mTOR by rapamycin may be a potentially effective therapeutic approach for OA."

Therapeutic significance

An oral dose of rapamycin, 3-6 mg, once a week is comparable to the systemic dose used in the mouse model. This study suggests that rapamycin may reduce human osteoarthritis, especially of knee joint by the mechanism of reduction of inflammation and increase in autophagy and thus maintaining the chondrocyte cells and the cartilage extracellular matrix.

I. OSTEOARTHRITIS

Introduction to Osteoarthritis

At one time, it was thought that Osteoarthritis was not a real disease; but just the natural result of "wear and tear". Osteoarthritis is due to loss of the articular cartilage that covers the bones in the joint. Just like the rubber on an automobile tire wears out; so to it was thought the the cartilage wears out. However, unlike the rubber in a tire, the cartilage is maintained by living cells, chondrocytes. Osteoarthritis is due to failure of the chondrocytes, the only cell in cartilage, to properly do their job, maintain the cartilage


Aging is the greatest risk factor for osteoarthritis. Two of the "usual suspects" play a major role in the pathogenesis of aging. These are a decrease in autophagy and an increase in inflammation. Both are related to an increase activity of mTOR. Furthermore, research studies on animal models of osteoarthritis have shown that use of rapamycin to lower the activity of mTOR ameliorates the disease process. In this discussion, we will take a close look at a number of very excellent studies showing the role of mTOR increasing OA and rapamycin in decreasing OA.  


The experimental studies are of 3 types:

I. Mouse model with systemic rapamycin.

II. Mouse model with intra-articular rapamycin.

III. In Vitro studies, generally using human cartilage cells from osteoarthritis patients.


Some studies were done to show the role of autophagy and other studies were done to show role of inflammation. Both autophagy and inflammation are very important in the pathogenesis of osteoarthritis. These  various studies combined, provide an extraordinary picture of osteoarthritis on the cellular and molecular level. 


 

Epidemiology of Osteoarthritis

"Arthritis is the most common cause of disability among U.S. adults" (1). A 2010-2012 study found that 52.5 million (22.7%) of adult aged >18 years had doctor-diagnosed arthritis..."Among persons with heart disease, diabetes, and obesity; the prevalence of doctor-diagnosed arthritis were 49.0%, 47.3%, and 31.2%, respectively" (1).


                                                                      Prevalence Osteoarthritis

                                                        % population                    % with Osteoarthritis

Heart Disease                                  11.5%                                      49.0%

Diabetes                                             9.0%                                       47.3%

Obesity                                              28.2%                                      31.2%


Association BMI and OA HIP, Knees, and Hand general population, adjusted for age, variables


                                               Hip OA                   Knee OA                   Hand OA

Body mass

Normal 20-25                          1                             1                                1

Overweight 26-30                  0.82                       2.02                         0.98

Obese >30                               1.11                       2.81                         2.59


Heart disease, Diabetes and Obesity are the most common age-related diseases associated with elevated activity of mTOR and have same increased risk as osteoarthritis.


As regards specific joints, there is no increased risk with weight for hip osteoarthritis. Osteoarthritis knee is associated with both overweight and obesity. Osteoarthritis hands is associated with obesity suggesting an elevated mTOR role. 


This data suggested that for persons of osteoarthritis of knees and possible hands reduction of obesity and reduction of elevated mTOR  is most likely be helpful.


Other studies have shown major impact of injury and proper joint alignment. Occupational joint loading is risk factor.   Moderate levels of physical activity have not been linked to increased osteoarthritis risk. 


As regards physical activity, those meeting recommendations had adjusted risk of 18.6% compared to persons classified as inactive activity which had adjusted risk of 24.0%. The decrease risk with increased physical activity is consistent with osteoarthritis being an age-related, increased mTOR type disease.


Another study (2) confirmed that obesity is a most important risk factor for osteoarthritis of knees, a weak risk factor for osteoarthritis of hands and not a risk factor for osteoarthritis of hips. 




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III Mouse Model OA, intra-articular rapamycin

Local intra-articular injection of rapamycin delays articular cartilage degeneration mouse model OA

Takayama, Pittsburgh, PA, Kobe, Japan, 2014.

Abstract:

Introduction: "Recent studies have revealed that rapamycin activates autophagy in human chondrocytes preventing the development of OA like changes in vitro, while the systemic injection of rapamycin reduces severity of experimental OA in a murine model of OA in vivo...The goal of current study was to examine the beneficial effects of local intra-articular injecton of rapamycin in a murine model of OA and to elucidate the mechanism of action of rapamycin on articular cartilage."


Methods: Destabilization of the medial meniscus (DMM) was performed in 10- week-old male mice to induce OA. Intra-articular injections of rapamycin (10uM) were administered twice weekly for 8 weeks. Articular cartilage damage was analyzed  by histology"...at 8 and 12 weeks after surgery...and immunohistochemistry.


Results: Intra-articular injection of rapamycin significantly reduced the severity of articular cartilage degradation at 8 and 12 weeks after DMM surgery. A reduction in mTOR expression and the activation of LC3 (an autophagy marker) in the chondrocytes was observed in the rapamycin treated mice. Rapamycin treatment also reduced VEGF, COL10A1, MMP13 at 8 and 12 weeks after DMM surgery."


Conclusion: These results demonstrate that the intra-articular injection of rapamycin could reduce mTOR expression, leading to a delay in articular cartilage degradation in our OA murine model. Our observations suggest that local-intra-articular injection of rapamycin could represent a potential therpapeutic approach to prevent OA."

Results

1. Local intra-articular injection of rapamycin delayed cartilage degradation. This was demonstrated by histologic exam.


2. Intra-articular injection of rapamycin decreased mTOR and increased LC3 expression. LC3 is a marker of increased autophagy.  LC3-positive articular cartilage cels maintained more proteoglycan staining [good].


3. The intra-articular injection of rapamycin decreased VEGF (vascular endothelial growth factor) expression in articular cartilage. VEGF is related to development of OA. 


4. The local intra-articular injection of rapamycin decreased CoL10A1-positive cells [good] and MMP13 expression [good]. [they are markers of chondrocyte hypertrophy, bad].



Discussion

In discussion and diagram they propose 3 mechanisms by which rapamycin can have a beneficial effect on OA.

a. Autophagy. Rapamycin inhibits mTOR which promotes autophagy  which promotes decreases in apoptosis and results in cell survival.

b. VEGF. Decreasing mTOR decreases VEGF, which promotes MMP13 and Col10A1 both of which promote OA.

3. Akt pathway. Decrease in mTOR can increase Akt pathway by negative feedback and Akt promotes proteoglycan synthesis [good]. 

[Note: while intra-articular levels might increase Akt, oral intermittent rapamycin in humans will be used at level not to promote Akt.]


The two major beneficial pathways documnted in this study was increase in autophagy and decrease in VEGF.


"The results of the current study are the first to demonstrate that articular cartilage degeneration occurring after DMM surgery (cutting meniscus) correlates with increased mTOR expression, and consequently local intra-articular injection of rapamycin reduced mTOR expression, which delayed articular cartilage degradation.  Recently, systemic injection of rapamycin has been shown to reduce severity of OA in an experimental murine model."


"In this study, we demonstrated that after surgically induced joint instability, chondrocytes in the articular cartilage displayed increased mTOR expression during the progression of OA, suggesting that the activation of mTOR leads to articular cartilage degeneration. Furthermore, the inhibition of mTOR with rapamycin promoted a significant delay in progression of OA changes."


The study demonstrated that rapamycin increased autophagy as shown by LC3-expressing cells which decreased degeneration of articular cartilage.


The study also focused on role of VEGF and angiogenesis in promoting articular cartilage degeneration. The study showed decrease in VEGF was beneficial. 


VEGF promotes chondrocyte hypertrophy.  Chondrocyte hypertrophic-like changes contribute to the progression of early and late stages of OA. Hypertrophic-like changes promote calcification of extracellular matrix. COL10A1 and MMP13 are the major markers of hypertrophic chondrocytes. Rapamycin reduced VEGF causing reduction of COL10A1 and MMP13 and reduction of harmful hypertrophic changes in articular cartilage. 


[Note: The response of cartilage cells to the stress of surgical destabilization of joint appears to be a programmed response to increase mTOR. Unfortunately, the increased level of mTOR has a harmful effect and promotes degeneration of cartilage. The beneficial effect of rapamycin is reducing mTOR.]

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