Pelvic organ prolapse (POP) is the downward descent of one or more pelvic organs causing discomfort and reducing quality of life for many women [1]. The most common risk factors contributing to POP are vaginal delivery of children and advanced age. Elastic matrix is an important component of structural integrity in pelvic organs.  Studies have shown abnormal elastic matrix homeostasis in prolapsed tissues of both animal models and human subjects. However, there are currently no treatment options for POP to repair and regenerate damaged elastic matrix.  The lysyl oxidase-like 1 (LOXL1) knockout (KO) mouse model develops POP and mirrors the risk factors and incidence of POP in women [2]. It can therefore be used to assess the pathophysiology of POP and structural changes of the elastic matrix.
Nanoparticles (NPs) can be used as drug delivery vehicles to repair elastic matrix through controlled delivery of elastogenic factors. Previous studies have shown that transforming growth factor-beta1 (TGF-β1) promotes lysyl oxidase (LOX)-mediated crosslinking and stimulates elastogenesis [3]. Doxycycline (DOX) has been shown to augment elastic fiber repair by inhibiting matrix metalloproteinase-2 (MMP2) and the C-Jun-terminal kinase (JNK) pathway responsible for attenuating TGF-β1 expression [3]. NPs used to encapsulate TGF-β1 were prepared from polyethylene glycol-polylactic-co-glycolic acid (PEG-PLGA) copolymer, via a double-emulsion solvent evaporation method, using didodecyldimethylammonium bromide as a cationic amphiphilic stabilizer [3]. NPs used in this study were designed to have low toxicity with controlled TGF-β1 release profile, while the cationic stabilizers on the NP surface were expected to enhance TGF-β1-independent regenerative outcomes at sites of matrix disruption. We hypothesized that the use of TGF-β1 loaded PEG-PLGA NPs and exogenous DOX could upregulate pro-elastogenic protein expression and repair damaged elastic matrix. To test this hypothesis, non-epithelial vaginal cells (NEVCs) from LOXL1 KO mice were treated in vitro with TGF-β1 loaded NPs and exogenous DOX. Expression of proteins involved in maintaining elastin homeostasis was assessed.

NEVCs were dissected from nine single delivery non-prolapsed (PNP) and nine nulliparous (N) 20-week old mice to determine the difference in elastin homeostasis protein expression after delivery in the absence of prolapse. Each animal group had NEVCs isolated from three mice combined to create one pool (three pools total) and cultured until confluent. The cells were then seeded at 30,000 cells/well on a 6 well plate (n=6 replicate/animal) and cultured for 21 days. After 21 days, PNP cells served as untreated (control) or were treated with 1 µg TGF-β1 loaded 0.2 mg/ml NPs or 0.2 mg/ml blank NPs (without TGF-β1). 
The NEVCs from one multiparous prolapsed (MP) 30-week mouse were used to determine effects of DOX dose and exposure time on elastogenesis. NEVCs were seeded at 50,000 cells/well on a 6 well plate (n=6 replicate/animal), and treated with DOX. The NEVCs were treated for 24h with different DOX concentrations (0.01µg/ml, 0.1µg/ml, 1µg/ml, 10 µg/ml) and 1.1 µg/ml SP600125, a JNK inhibitor, to determine the DOX dose that would significantly enhance expression of elastin homeostasis proteins, including LOX isoforms, MMPs, tissue inhibitors of MMPs (TIMPs), JNK isoforms, extracellular signal-regulated kinase (ERK), and Fibulin 5 (Fib5). The most useful DOX concentration (1µg/ml) was then used to find the optimal time duration (0.5, 1, 2h) for upregulation of elastin homeostasis proteins. Finally NEVCs were treated with different concentrations of DOX at the optimal time duration (2h).  
Following the treatments, cell lysates were collected and assessed using western blot. Each sample was loaded into a 10% sodium dodecysulfate polyacrylamide gel electrophoresis. The gels were immunolabeled with primary antibodies for respective proteins followed by secondary antibodies for detection by fluorescence (Table 1). Fluorescence intensity of the protein bands was quantified and normalized relative to their respective β-actin band (exogenous loading control).  Experimental groups were further normalized to mean of respective controls. A t-test with Mann-Whitney rank sum posthoc test was used to determine statistically significant differences between elastin homeostasis protein expression in NEVC cells from PNP mice vs NEVCs from nulliparous mice, as well as and NEVC cells from PNP mice treated with TGF-β1 NPs (p<0.05) vs. untreated cells. One-way ANOVA with Bonferroni corrections (p<0.05) was used to assess statistically significant differences in the TGF-β1 NPs (pool 2&3) and DOX treated groups to their respective controls. For one-way ANOVA tests, when stated otherwise Dunn’s method was used to determine statistically significant differences between groups (p<0.05). Results are presented as mean ± standard error of the mean.

NEVCs from PNP mice had significantly decreased normalized expression of LOX 28kD (0.59±0.03), TIMP1 (0.57 ±0.12), TIMP4 (0.36±0.05) and significantly increased expression of MMP2 (1.9 ±0.26), compared to NEVCs from N mice (LOX 28kD: 1.0±0.04; TIMP1: 1.0±0.06; TIMP4: 1.0±0.08; MMP2: 1.0±0.04). This suggests that vaginal wall expression of pro-elastogenic proteins is decreased and proteolytic enzymes increased following vaginal delivery. Treatment of PNP NEVCs with TGF-β1 NPs significantly increased LOX 50kD expression (0.77 ± 0.07) and decreased MMP2 expression (0.52 ± 0.06) compared to untreated cultures (LOX: 1.0 ± 0.09, MMP2: 1.0 ± 0.11). This suggests that TGF-β1 NPs upregulate LOX mediated crosslinking and decrease MMP2, improving structural integrity of the elastic matrix.
Treatment of NEVCs with 1µg/ml DOX significantly increased JNK 48kD (1.2±0.04) and JNK 43+48kD (1.2±0.05), and TIMP2 24kD (2.2±0.33) compared to control untreated NEVCs (JNK 48kD: 1.0±0.07; JNK 43+48kD: 1.0±0.07, and TIMP2 24kD: 1.0±0.18) and significantly decreased the MMP2/TIMP2 ratio (Dunn’s Method, 0.41±0.07) compared to controls (1.0±0.20). The decrease in MMP2/TIMP2 ratio suggests that 1µg/ml DOX promotes an increase in pro-elastogenic proteins. When MP NEVCs were treated with different concentrations of DOX for 2h, there was a significant decrease in MMP2, MMP2/TIMP2, LOX 28kD, ERK 40kD, and ERK 43kD compared to the untreated group (Figure 1). There was a significant increase in TIMP2 68kD and Fib5 (Dunn’s method) compared to the untreated group (Figure 1). These results show that treating the cells with DOX results in upregulation of pro-elastogenic proteins and downregulation of matrix degenerative proteins, suggesting improved structural integrity of the elastic matrix.

The decreased expression of pro-elastogenic proteins in untreated PNP NEVC cultures compared to nulliparous NEVC cultures suggest degradation of elastic matrix in the LOXL1 KO mouse model even in the absence of prolapse. Expression of pro-elastogenic proteins was increased and that of MMPs decreased in PNP NEVCs upon treatment with TGF-β1 NPs, demonstrating improved elastin homeostasis, confirming our hypothesis and suggesting their therapeutic potential. Similar results were observed with MP NEVCs treated with DOX.

This study demonstrates the pro-elastogenic effects of TGF-β1 and DOX to repair damaged elastic matrix in POP. These findings suggest that TGF-β1 NPs can be used as a regenerative therapy for POP and DOX could be investigated to determine possible pro-elastogenic effects and clinical applications.

Jelovsek JE, Maher C, Barber MD. Pelvic organ prolapse. Lancet. 2007;369(9566):1027-1038. doi:10.1016/S0140-6736(07)60462-0
Dahal S, Kuang M, Rietsch A, Butler RS, Ramamurthi A, Damaser MS. Quantitative Morphometry of Elastic Fibers in Pelvic Organ Prolapse [published online ahead of print, 2021 Mar 23]. Ann Biomed Eng. 2021;10.1007/s10439-021-02760-9. doi:10.1007/s10439-021-02760-9
Camardo A, Seshadri D, Broekelmann T, Mecham R, Ramamurthi A. Multifunctional, JNK-inhibiting nanotherapeutics for augmented elastic matrix regenerative repair in aortic aneurysms. Drug Deliv Transl Res. 2018;8(4):964-984. doi:10.1007/s13346-017-0419-y