The mechanisms and potential of stem cell therapy for penile fibrosis

[madman]

Abstract

Fibrosis is often caused by chronic tissue injury leading to a persisting inflammatory response with excessive accumulation of extracellular connective tissue proteins. Peyronie’s disease, urethral stricture, and penile (corpora cavernosa) fibrosis are localized fibrotic disorders of the penile connective tissues that can substantially impair a patient’s quality of life. Research over the past few decades has revealed the ability of stem cells to secrete a wide range of paracrine factors, a characteristic that could be exploited therapeutically to prevent and treat several inflammatory and fibrotic diseases. In preclinical studies, mesenchymal stem cells (MSCs) have proven to be the most effective and readily available type of stem cells for therapeutic use. An important advantage of MSCs is their ability to circumvent the immune system and function as immunomodulatory ‘drug stores’ to influence multiple cell types simultaneously. Many studies using stem cells have been applied exclusively to corpora cavernosa fibrosis owing to its well-established disease models. A plethora of preclinical data suggests the benefit of stem cells for use in penile fibrosis. However, their exact mechanism of action and optimal timing and mode of administration must be determined before clinical translation.




Fibrosis is a wound-healing disorder that often occurs in response to chronic tissue injury and that is defined by a persisting inflammatory reaction and the subsequent excessive accumulation of extracellular connective tissue proteins such as collagen, elastin, and fibronectin (collectively termed the extracellular matrix (ECM))1,2. Typically, inflammation and ECM aggregation is an essential and reversible phase of the normal wound-healing process3. However, if the initial injury (for example, infection, mechanical stress, or autoimmune reaction) is not resolved in a timely manner or the wound-healing process itself becomes deregulated, this phase can gradually evolve into a permanent fibrotic reaction and lead to fibrogenesis, a process termed ‘fibrosis’4. Importantly, fibrosis is the conclusive pathological consequence of many chronic inflammatory disorders and can lead to a progressive loss of tissue and/or organ function5.

Localized fibrotic disorders of the penile connective tissues such as Peyronie’s disease6–8 and urethral stricture disease9 are thought to occur as a result of impaired wound healing. Peyronie’s disease is considered to be caused by lesions in the tunica albuginea following intercourse-related repetitive microtrauma caused by buckling of the penis, whereas urethral strictures can be caused by trauma to the urethra as a result of instrumentation (iatrogenic: bladder catheters) and/or infectious and inflammatory disorders (for example, sexually transmitted diseases)8,9. Penile fibrosis occurs as a diffuse fibrotic process in the corpora cavernosa of the penis and is the result of various conditions associated with erectile dysfunction (ED) such as diabetes mellitus, atherosclerosis, iatrogenic pelvic nerve damage (after radical prostatectomy for prostate cancer), and even aging-related ED8. Additionally, severe corporal fibrosis can occur acutely, in which case it is frequently the result of episodes of ischaemic priapism10. These fibrotic disorders can severely decrease the quality of life of patients by causing lower urinary tract symptoms (LUTS), the onset or worsening of ED, painful and deformed erections, major depressive disorders, and relationship issues11,12

At present, very few therapeutic strategies are available for the treatment of fibrotic conditions; however, the search for novel treatments has led to the discovery of the immunomodulatory capacities of stem cells. Stem cells are well known for their ability for self-renewal and differentiation into a diverse set of mature cell populations12. Moreover, the secretion of a wide range of paracrine factors, including growth factors, cytokines, chemokines, and even functional small RNAs (via extracellular vesicles), makes stem cells appealing for therapeutic application. These secreted factors enable stem cells to influence and modify their host environment, particularly during and early after tissue injury13–15. In recognition of these unique properties, a growing body of (preclinical) evidence has demonstrated the potential therapeutic role of stem cells in alleviating fibrosis16–20. Mesenchymal stem cells (MSCs) have been commonly used in this therapeutic context and have been shown to have a role in reducing fibrosis in animal models of lung21,22, liver23–25, kidney26–28, heart29,30, corpus spongiosum and urethra31, corpus cavernosum32,33, and tunica albuginea34 fibrosis. As of September 2018, ~60 clinical trials (active or recruiting) are evaluating the efficacy of MSCs for the treatment of various fibrotic disorders (for example, hepatic, Crohn’s disease-related intestinal, cardiac, and pulmonary fibrosis).

The precise mechanisms governing the antifibrotic properties of MSC therapy are yet to be elucidated. However, the leading theory is that MSCs function as ‘drug-stores, influencing several cell types (for example, cells from the innate and adaptive immune system, resident fibroblasts, and smooth muscle cells) and the production of several profibrotic and antifibrotic factors simultaneously12. Most preclinical studies suggest that MSCs exert their antifibrotic functions through immunomodulation, thereby limiting the host’s response to injury and preventing the onset of fibrosis12,15. Another putative mechanism is the attenuation of profibrotic phenotypic changes of resident fibroblasts into the contractile and ECM-producing myofibroblasts35. Furthermore, stem cells can directly modulate ECM composition on the basis of their ability to secrete high levels of matrix metalloproteinases (MMPs) and other matrix-modulating enzymes (for example, through inhibition of tissue inhibitors of metalloproteinases (TIMPs))15. Nonetheless, these hypotheses have not yet been proven and additional studies focusing on the mechanisms of MSC function are ongoing12,15.

In the past decade, stem cells have also been evaluated for the prevention and treatment of fibrosis of the male genitourinary tract (Peyronie’s disease, urethral stricture, and corpora cavernosa fibrosis). In this Review, we provide an overview of current research on stem cells for the treatment of penile fibrosis, with an emphasis on the specific mechanisms of antifibrotic activity




*Pathophysiology of fibrotic disorder

*Stem cells

*MSCs
MSC differentiation and secretome

*Stem cell therapy for penile fibrosis

*Corpora cavernosa fibrosis
Stem cell therapy for corpora cavernosa fibrosis

*Peyronie’s disease
Stem cell therapy for Peyronie’s disease

*Urethral stricture
Stem cell therapy for urethral stricture




Conclusions

To date, the clinical application and investigation of conventional antifibrotic therapies have yielded limited results. Conventional approaches focus on the inhibition of one small cog in the large machinery of fibrosis, resulting in the activation of auxiliary pathways that counteract the effects of these antifibrotic drugs. However, the use of stem cells in translational research has the potential to exert antifibrotic functions on several levels by modulating the host response. Despite the amount of research on stem cells and penile fibrosis, the field is still in its infancy and is subject to many limitations. Most preclinical research regarding stem cells in penile fibrosis has focused on corpora cavernosa fibrosis owing to its clear pathophysiology (iatrogenic postprostatectomy ED and corpora cavernosa fibrosis) and representative animal models. Conversely, Peyronie’s disease and urethral stricture disease research have been limited by poor disease models and unvalidated findings. Thus, the treatment of corpora cavernosa fibrosis with stem cells seems to be the closest to potential clinical application given additional studies evaluating the efficacy, dosage, timing, and route of administration of stem cells or SVF.

 

[madman]

Fig. 1 | Overview of the wound-healing process and fibrosis. a | During an acute tissue insult, innate immune cells are recruited to restore homeostasis via normal wound healing. During the haemostasis phase, tissue damage activates the coagulation cascade and induces platelet activation and blood clot formation. When the epithelium is damaged, prestored interleukin-1 (IL-1) is released. Moreover, circulating platelets release coagulation factors for haemostasis (fibrin activation); and growth factors, such as platelet-derived growth factor (PDGF), a potent chemoattractant for inflammatory cells and mitogenic factor for myofibroblasts, and transforming growth factor-β1 (TGFβ1), which stimulates extracellular matrix (ECM) production and fibroblast-to-myofibroblast transformation1,38,165. Aside from its ubiquity in the coagulation cascade, thrombin also activates C-C motif chemokine 2 (CCL2)4 , and myeloid cells (macrophages) migrate from the bone marrow to the site of injury in response to the CCL2 gradient. Together with neutrophils, these cells release a wide array of toxic mediators — reactive oxygen species (ROS) and reactive nitrogen species (RNS) — that exacerbate tissue damage if they are not removed in a timely manner166. b | The inflammation phase is characterized by the chemotaxis and activation of innate immune cells by CCL2, IL-1, PDGF and TGFβ1. The classic inflammatory macrophage exhibits a strong microbicidal or tumoricidal activity through expression of IL-1β, tumour necrosis factor (TNF) and IL-6 (REFS70,167). Additionally, macrophages also produce large quantities of TGFβ1, which has an important role in myofibroblast generation87. Local inflammation activates naive (unactivated) T cells of the adaptive immune system towards different phenotypes via various cytokines2. Precursor B cells are activated mainly by T cell-dependent cytokines and antigen presentation (although T cell-independent pathways also exist). A fully matured B cell becomes a nondividing plasma cell, producing large quantities of antibodies directed against a specific antigen168. c | During the proliferation phase, the immune system — via secretion of cytokines, chemokines and free radicals — attempts to remove the triggering factor while activating the myofibroblasts that regulate angiogenesis and ECM production in normal wound healing169. In fibrotic conditions, dysregulation of genes involved in ECM remodelling occurs; for example, downregulation of matrix metalloproteinases (MMPs; which remove excessive collagen fibres) and upregulation of tissue-inhibitors of metalloproteinases (TIMPs; produced by myofibroblasts and macrophages)170–172. d | During the remodelling phase, activated myofibroblasts promote wound contraction. Different collagen types (switch from type III to type I collagen) are produced, blood vessels repaired, excessive scar tissue removed, and epithelial cells divide and migrate over the basal layers to regenerate the epithelium, restoring the damaged tissue to its normal appearance with minimal scar tissue2,37. Failure to effectively eliminate the triggering factors can induce persistent inflammation of the tissue, which leads to fibrosis. A few crucial concepts are generally applicable for the progression of fibrotic disease: persistent chronic tissue injury owing to chronic exposure and/or inflammation; ongoing recruitment of innate and adaptive immune cells to create a profibrogenic environment favouring chronic inflammation, and inefficient remodelling modulated by tissue hypoxia and subsequent neoangiogenesis173,174. TSLP, thymic stromal lymphopoietin. TH cell, T helper cell; Treg cell, regulatory T cell; VEGF, vascular endothelial growth factor

 

[madman]

Fig. 2 | Penile fibrosis. Peyronie’s disease, urethral stricture, and corpora cavernosa fibrosis are fibrotic disorders of the penile connective tissues that are characterized by changes in the collagen composition (formation of a fibrous plaque) of the tunica albuginea, the urethral corpus spongiosum, and the penile corpora cavernosa, respectively8,11. Corpora cavernosa fibrosis is characterized by generalized fibrosis in the erectile tissue of the penis. When the expansion of corporal spongy tissue is compromised as a result of fibrosis, rapid filling of the sinusoids will no longer occur, leading to a subsequent lack of compression of the subtunical venules94, rendering the penis incapable of becoming completely rigid (corporal Veno-occlusive dysfunction). The most important aetiological factors for corpora cavernosa fibrosis include atherosclerosis (owing to chronic penile hypoxia), diabetes mellitus (owing to endothelial dysfunction, nerve damage, impaired arterial flow, reactive oxygen species and advanced glycation end-products (AGEs)), and iatrogenic causes (damage of the neurovascular bundle during prostatectomy in patients with prostate cancer)94–96. Peyronie’s disease is a localized fibrotic disorder situated on the tunica albuginea of the penis. Peyronie’s disease is associated with erectile dysfunction (veno-occlusive dysfunction), curvatures of up to 90° or more (which impair sexual intercourse) and up to 50% of patients experience clinically significant depression and/or relationship problems175. The leading hypothesis states that Peyronie’s disease is caused by repetitive microtrauma as a result of buckling of the penis during intercourse176. Patients experience an acute phase with a painful penis (usually during erections), no palpable plaque, and a progressive curvature. After 12–24 months, the disease evolves into a chronic, stable plaque and curvature that is painless. Currently, available treatment options include surgery and intratunical injections of collagenase in stable disease. Other (medical) treatment options have proven unsuccessful177. Urethral strictures are characterized by an abnormal narrowing of the urethra that functionally causes a bladder outlet disorder, which can negatively affect a patient’s quality of life by leading to genitourinary infection (epididymitis, prostatitis, cystitis, or pyelonephritis), impairing voiding and causing secondary bladder overactivity (with complaints of urgency and urgency incontinence)9. Several aetiologies have been proposed and are categorized as iatrogenic, traumatic, inflammatory, and idiopathic9. Since the advent of endourological surgical techniques and urine catheters, the iatrogenic factors have been the most important causes of urethral strictures

 

[madman]

Fig. 3 | Pro-inflammatory and anti-inflammatory MSC phenotypes. Mesenchymal stem cells (MSCs) can both promote and inhibit fibrosis, depending on the inflammatory context in which they function35. This paradox is in part explained by their broad immunoregulatory capacities with respect to both the adaptive and innate immune system85. By interacting with cells of the innate immune system, MSCs can elicit both anti-inflammatory and pro-inflammatory effects35. MSCs are predominantly in a state of quiescence and reduced cellular metabolism47. This quiescent state seems to be necessary for the long-term preservation of the reconstituting capacity of stem cells. Stem cells can exit quiescence and rapidly expand and differentiate in response to stress35. In the absence of inflammation (low tumor necrosis factor (TNF) and interferon (IFN)-γ levels), MSCs can adopt a pro-inflammatory phenotype and enhance M1 macrophage polarization and T cell response by secreting pro-inflammatory cytokines (IL-1, IL-2, TNF, and IFNγ) 35,85. Secretion of TGFβ1 induces the myofibroblast phenotype, further establishing the inflammatory and profibrotic milieu. In the presence of inflammation (high TNF and IFNγ levels), MSCs are activated and adopt an immunosuppressive phenotype35. Activated MSCs can also skew the differentiation of monocytes towards an anti-inflammatory and antifibrotic profile, therefore, shifting the balance towards anti-inflammatory M2 macrophage polarization rather than pro-inflammatory M1 macrophage polarization.

 

[madman]

Fig. 4 | The origin of myofibroblasts. Myofibroblasts are possibly the most unique cells involved in the wound-healing process; they actively regulate the extracellular matrix (ECM) while simultaneously possessing a cytoskeletal contractile apparatus similar to that of smooth muscle cells178,179. Local cells undergo a phenotypic switch into myofibroblasts, which is indicated by the production of the myofibroblast marker α-smooth muscle actin (αSMA) and ECM components such as collagens (mostly type I and III collagen), laminin, and fibronectin, a process that is mainly driven by transforming growth factor-β1 (TGFβ1)170–172. The principal myofibroblast progenitor after the tissue injury seems to vary, and includes epithelial or endothelial cells, which undergo epithelialto-mesenchymal transition (EMT) or endothelial-to-mesenchymal transition (EndoMT), respectively; local tissue-residing fibroblasts (within the tissue where the fibrosis occurs); circulating bone marrow-derived fibrocytes; tissue-resident mesenchymal stem cells (MSCs); and smooth muscle cells. However, the relative contribution of each cell type to the formation of myofibroblasts in a fibrotic scar is still under debate180. For EMT and EndoMT, the organized dedifferentiation of epithelial cells occurs with a loss of polarity, adherence, and tight junctions through a process that is mainly driven by TGFβ1, fibroblast growth factor 2 (FGF2), insulin-like growth factor 2 (IGF2), and epidermal growth factor (EGF)181–183. Almost 0.5% of the leukocytes in peripheral blood are fibrocytes, which can be identified by the expression of the haemotpoeitic cell marker CD34, leukocyte markers (CD11b, CD13, and CD45), and fibroblast products (such as type I and III collagen and fibronectin); however, fibrocytes lack specific macrophage, dendritic cell, or B cell markers. TGFβ1 and endothelin-1 stimulate the activation and differentiation of fibrocytes into myofibroblast-like cells (αSMA-positive)180. Tissue-resident MSCs can be distinguished from fibrocytes by their lack of haemotpoeitic and leukocyte marker expression and αSMA positivity15,180. During atheromatous plaque formation, smooth muscle cells have been shown to dedifferentiate into myofibroblasts and are considered to be an important contributor to the associated fibrosis154.

 

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