Outgrowth of chondrocytes from human articular cartilage explants and expression of α-smooth muscle actin

The objectives of this study were to investigate the effect of various enzymatic treatments on the outgrowth of chondrocytes from explants of adult human articular cartilage and the expression of a specific contractile protein isoform, alpha-smooth muscle actin, known to facilitate wound closure in other connective tissues. Explants of articular cartilage were prepared from specimens obtained from patients undergoing total joint arthroplasty. The time to cell outgrowth in vitro was determined and the expression of alpha-smooth muscle actin shown by immunohistochemistry. Treatment of the explants with collagenase for 15 minutes reduced the time to outgrowth from more than 30 days to 3 days. Hyaluronidase, chondroitinase ABC, and trypsin applied for the 15-minute period had no effect on the time to cell outgrowth when compared with untreated controls. Pretreatment with hyaluronidase prior to collagenase reduced the time to outgrowth. A notable finding of this study was that the majority of chondrocytes in the adult human articular cartilage specimens and virtually all of the outgrowing cells contained alpha-smooth muscle actin. We conclude that human articular chondrocytes have the capability to migrate through enzymatically degraded matrix and express a contractile actin isoform. Collagenase treatment reduces the time required for cell outgrowth.     

Expression of smooth muscle actin in connective tissue cells participating in fracture healing in a murine model

The role of alpha-smooth muscle actin (SMA)-expressing fibroblasts in the contraction of skin wounds has been known for three decades. Recent studies have demonstrated that osteoblasts can also express the gene for this contractile muscle actin isoform and can contract a collagen-glycosaminoglycan analog of extracellular matrix in vitro. These findings provided rationale for the hypothesis that SMA-expressing cells contribute to fracture healing by drawing the bone ends together. To begin to test this hypothesis, immunohistochemistry was employed to evaluate the distribution of connective tissue cells expressing SMA in a mouse model of successful fracture healing. The results demonstrated that the majority of the cells comprising the mesenchymal tissue interposed between the fracture ends contained SMA after 7 and 21 days, supporting the working hypothesis. Most of the osteoblasts lining the surfaces of newly forming bone and the chondrocytes comprising the cartilaginous callus also expressed this contractile actin isoform. The maximal SMA expression extended from 7 to 21 days postfracture. The finding of high levels of SMA expression in connective tissue cells participating in fracture healing suggests that SMA-enabled contraction may be playing a role in the healing process. These results warrant further study of the specific SMA-dependent cell behavior.     

Smooth muscle actin expression by human articular chondrocytes and their contraction of a collagen-glycosaminoglycan matrix in vitro.

Recent studies have demonstrated that human articular chondrocytes can express the gene for a contractile muscle actin, alpha-smooth muscle actin (SMA), in situ. One objective of this work was to evaluate the SMA-content of isolated human articular chondrocytes using Western blot analysis and to correlate the amount of SMA in the cells with passage number and the number of days in culture. A second objective was to determine if articular cartilage-derived cells expressing the gene for SMA in vitro also continue to express type II collagen. A final aim of the current study was to determine if SMA-containing cartilage-derived cells were capable of contracting a collagen glycosaminoglycan analog of extracellular matrix in vitro. Articular chondrocytes were isolated from 13 patients undergoing total joint arthroplasty. Cells were serially passaged through passage 7. Samples were allocated for Western blot analysis of SMA. Cells in monolayer culture were also stained immunohistochemically for SMA and type II collagen. Cells from passage 3 and 7 were seeded into a porous type I collagen-glycosaminoglycan matrix and the diameter of the scaffolds measured every other day for 21 days. Immunohistochemistry of the articular cartilage samples revealed SMA in the articular chondrocytes in situ with a greater percentage of cells staining positive in the superficial half (60 +/- 1.2%; mean +/- SEM) of the cartilage than in the basal half (28 +/- 1.3%). There was an increasing amount of SMA in the cells in monolayer culture with passage number and a meaningful correlation of the SMA content with the days in culture (linear regression analysis; R2 = 0.72). Double staining for SMA and type II collagen showed that type II collagen-expressing cells in monolayer could also express SMA. SMA-containing cells were found to contract the collagen glycosaminoglycan matrix, with the cells containing more SMA (passage 7 cells) displaying more matrix contraction than those with a lesser amount of SMA (passage 3 cells). The results indicate that control of the expression of SMA may be important when employing articular chondrocytes, expanded in monolayer culture, for implantation alone or in a cell-seeded matrix for cartilage repair procedures.     

Musculoskeletal connective tissue cells with muscle: Expression of muscle actin in and contraction of fibroblasts, chondrocytes, and osteoblasts

The expression of the gene for a muscle actin in certain nonmuscle cells and the contraction of these connective tissue cells has been associated with several important physiological and pathological processes; the contraction of healing skin wounds and the contracture in Dupuytren's disease being two notable examples. Studies in recent years have shown that a much wider variety of connective tissue cells than previously considered, including cells in many of the musculoskeletal tissues, e.g., chondrocytes and osteoblasts, can express the gene for alpha-smooth muscle actin and can display contractile behavior. These findings suggest that muscle actin-enabled cell contraction may also be playing important roles in the other connective tissues comprising the musculoskeletal system, namely, tendon, ligament, meniscus, intervertebral disc, articular cartilage, and bone.     

Use of an alpha-smooth muscle actin GFP reporter to identify an osteoprogenitor population

Identification of a reliable marker of skeletal precursor cells within calcified and soft tissues remains a major challenge for the field. To address this, we used a transgenic model in which osteoblasts can be eliminated by pharmacological treatment. Following osteoblast ablation a dramatic increase in a population of alpha-smooth muscle actin (alpha-SMA) positive cells was observed. During early recovery phase from ablation we have detected cells with the simultaneous expression of alpha-SMA and a preosteoblastic 3.6GFP marker, indicating the potential for transition of alpha-SMA(+) cells towards osteoprogenitor lineage. Utilizing alpha-SMAGFP transgene, alpha-SMAGFP(+) positive cells were detected in the microvasculature and in the osteoprogenitor population within bone marrow stromal cells. Osteogenic and adipogenic induction stimulated expression of bone and fat markers in the alpha-SMAGFP(+) population derived from bone marrow or adipose tissue. In adipose tissue, alpha-SMA(+) cells were localized within the smooth muscle cell layer and in pericytes. After in vitro expansion, alpha-SMA(+)/CD45(-)/Sca1(+) progenitors were highly enriched. Following cell sorting and transplantation of expanded pericyte/myofibroblast populations, donor-derived differentiated osteoblasts and new bone formation was detected. Our results show that cells with a pericyte/myofibroblast phenotype have the potential to differentiate into functional osteoblasts.     

Evidence that bone morphogenetic protein 4 has multiple biological functions during kidney and urinary tract development

BACKGROUND: We have suggested that bone morphogenetic protein 4 (BMP4), acting on the Wolffian duct and ureter epithelium, determines the budding site of the ureter by locally antagonizing ubiquitous inductive signal(s) from the metanephric mesenchyme. In the present study, we examine the effect of BMP4 on the development of metanephric and periureteral mesenchymal cells, which express the BMP type I receptor gene, Bmpr1a (Alk3). METHODS: Urogenital tissues obtained from Bmp4 heterozygous null mutant (Bmp4+/-) embryos at different stages, and metanephric and ureteral tissue explants cultured in the presence of recombinant BMP4 were subjected to morphologic, immunohistochemical and in situ hybridization analyses. To examine the chemotactic activity of BMP4 for periureteral mesenchymal cells, a modified Boyden chamber assay was performed. RESULTS: Many of the kidneys of newborn Bmp4+/- mice contained multicystic dysplastic regions. This morphology was preceded by abnormally high apoptotic activity in the metanephric mesenchyme of mutant embryos at E14.5. In whole metanephric explants, BMP4 uniformly promoted the expansion of the Pax2-negative and weakly Foxd1 (previously Bf2) -positive peripheral stromal compartment of metanephric mesenchyme in the presence of fibroblast growth factor 2 (FGF2). In addition, in isolated metanephric mesenchyme, BMP4-loaded beads prevented apoptosis locally. Thus, BMP4 prevents cell death and promotes the growth of the metanephric mesenchyme. The effect of BMP4 on periureteral mesenchyme is different from its effect on metanephric mesenchyme. In utero, periureteral mesenchymal cells condense around the ureter epithelium, followed by differentiation into smooth muscle cells at a site where Bmp4 is intensely expressed. Analysis of Bmp4+/- ureters at E15.5 reveals that the alpha-smooth muscle actin (alpha-SMA)-positive cells are low in number. In vitro, BMP4-loaded beads promote the accumulation of periureteral mesenchymal cells to form several cell layers surrounding the beads. In addition, in a Boyden chamber assay, BMP4 increases the migration of periureteral mesenchymal cells through the filter. Thus, BMP4 can serve as a chemoattractant for periureteral mesenchymal cells and induce locally the smooth muscle layer of the ureter at Bmp4-expressing sites. CONCLUSION: Depending on local context, BMP4 has several biological actions on the morphogenesis of different portions of the excretory system, namely, the development of the ureterovesical junction, the ureter, and the kidney.     

Histological changes in the human anterior cruciate ligament after rupture

BACKGROUND: Four phases in the response to injury of the ruptured human anterior cruciate ligament are observed histologically; these include an inflammatory phase, an epiligamentous repair phase, a proliferative phase, and a remodeling phase. One objective of this study was to describe the histological changes that occur in the ruptured human anterior cruciate ligament during these phases. Myofibroblast-like cells that contain alpha-smooth muscle actin are present in the midsubstance of the intact human anterior cruciate ligament. A second objective of this study was to determine whether an increased number of myofibroblast-like cells is found in the midsubstance of the ruptured human anterior cruciate ligament because it was thought that those cells might be responsible in part for the retraction of the ruptured anterior cruciate ligament. In the early phase of this study, it was found that the number of myofibroblast-like cells in the midsubstance of the ruptured anterior cruciate ligament was actually decreased, and this hypothesis was abandoned. During the epiligamentous repair phase, synovial tissue was formed that covered the ends of the ruptured anterior cruciate ligament. Most of the synovial lining cells were myofibroblast-like cells that contained alpha-smooth muscle actin. The primary objective of this study was to determine the location and the characteristics of the alpha-smooth muscle actin-containing myofibroblast-like cells that appear in the human anterior cruciate ligament following rupture. METHODS: Twenty-three ruptured and ten intact human anterior cruciate ligaments were evaluated for cellularity, nuclear morphology, blood vessel density, and percentage of cells containing a contractile actin isoform, alpha-smooth muscle actin. The histological features of the synovial and epiligamentous tissues were also described. RESULTS: At no time after rupture was there evidence of tissue-bridging between the femoral and tibial remnants of the anterior cruciate ligament. The ruptured ligaments demonstrated a time-dependent histological response, which consisted of inflammatory cell infiltration up to three weeks, gradual epiligamentous repair and resynovialization between three and eight weeks, and neovascularization and an increase in cell number density between eight and twenty weeks. Compared with the intact ligaments, there was a decrease in the percentage of myofibroblast-like cells containing alpha-smooth muscle actin within the remnant of the ligament. However, many of the epiligamentous and synovial cells encapsulating the remnants contained alpha-smooth muscle actin. CONCLUSIONS: After rupture, the human anterior cruciate ligament undergoes four histological phases, consisting of inflammation, epiligamentous regeneration, proliferation, and remodeling. The response to injury is similar to that reported in other dense connective tissues, with three exceptions: formation of an alpha-smooth muscle actin-expressing synovial cell layer on the surface of the ruptured ends, the lack of any tissue bridging the rupture site, and the presence of an epiligamentous reparative phase that lasts eight to twelve weeks. Other characteristics reported in healing dense connective tissue, such as fibroblast proliferation, expression of alpha-smooth muscle actin, and revascularization, also occur in the ruptured human anterior cruciate ligament. CLINICAL RELEVANCE: Unlike extra-articular ligaments that heal after injury, the human intra-articular anterior cruciate ligament forms a layer of synovial tissue over the ruptured surface, which may impede repair of the ligament. Moreover, a large number of cells in this synovial layer and in the epiligamentous tissue express the gene for a contractile actin isoform, alpha-smooth muscle actin, thus differentiating into myofibroblasts. These events may play a role in the retraction and lack of healing of the ruptured anterior cruciate ligament.     

Distribution of chondrocytes containing alpha-smooth muscle actin in human articular cartilage.

Relatively little is known about the contractile behavior of the human articular chondrocyte. Other connective tissue cells are known to express a contractile actin isoform, alpha-smooth muscle actin, in response to injury, at selected stages of wound healing, and in certain pathological conditions. This and recent work demonstrating contractile behavior in adult canine articular chondrocytes in vitro prompted the present study of the distribution of alpha-smooth muscle actin-containing chondrocytes in human articular cartilage. Approximately 75% of the chondrocytes in the superficial region of cartilage expressed alpha-smooth muscle actin as demonstrated by immunohistochemistry. In contrast, only approximately 10% of the cells in the deep region stained for this contractile actin isoform. There was no correlation of the percentage of alpha-smooth muscle actin-positive cells in either region with Mankin grade or with age. This is the first report of a contractile potential for human articular chondrocytes. The roles of alpha-smooth muscle actin in these cells warrant further investigation. The question of whether it is necessary to refer to these cells as myochondrocytes is considered.     

The presence of smooth muscle actin in fibroblasts in the torn human rotator cuff.

The rotator cuff frequently sustains athletic and occupational injury, often resulting in chronic pain and disability. However, despite the high incidence of such shoulder problems, the pathophysiology of rotator cuff injury and healing has not yet been fully elucidated. The notable finding of this study was the presence of a contractile actin isoform, alpha-smooth muscle actin (SMA), in nonvascular cells in all of the seven torn human rotator cuff specimens evaluated immunohistochemically. Up to 95% of cells in any one region, and over 95% of elongated cells found in association with crimped collagen, contained SMA. Most of the cells staining positive for SMA in these sections had morphological features of the fibroblast, though a small number were chondrocyte-like. Treatment of cells growing out from human rotator cuff explants with TGF-beta1 significantly increased the amount of SMA evaluated by Western blot analysis. PDGF-BB and IFN-gamma had no effect on the cell content of SMA. This is the first documentation of the presence of SMA-positive cells in the human rotator cuff tendon. SMA has been found in a number of other healing connective tissues including skin, ligament, meniscus, cartilage, and other types of tendon. Of importance are previous findings that SMA-positive cells can contract a collagen-glycosaminoglycan analog of extracellular matrix in vitro. The results of the present study thus suggest that SMA-containing cells could contribute to the retraction of the torn ends of a ruptured rotator cuff and play an important role in healing.     

人降钙素基因相关肽重组腺相关病毒在原代培养大鼠阴茎海绵体平滑肌细胞中的表达及其作用

目的:观察腺相关病毒载体介导的人降钙素基因相关肽(hCGRP)基因在原代培养的大鼠阴茎海绵体平滑肌(CCSM)细胞的表达及其作用,探讨其应用于勃起功能障碍(ED)基因治疗的可行性。方法:原代培养的SD大鼠CCSM细胞随机分为4组:实验组、空病毒组、示踪剂组和对照组,分别以可分泌表达hCGRP重组腺相关病毒VssH-GCMV-hCGRP、空病毒VssHGCMV和表达绿色荧光蛋白(GFP)重组腺相关病毒VssCMV-GFP进行体外转染或不予任何处理。采用斑点印迹试验检测培养液中的hCGRP和放射免疫法检测转染细胞中的环磷酸腺苷(cAMP)和环磷酸鸟苷(cGMP)水平,观察hCGRP和示踪剂GFP的表达及其对CCSM细胞的影响。结果:重组腺相关病毒载体能有效地将外源基因hCGRP导入大鼠CCSM细胞并稳定表达,与空病毒组和对照组相比,该病毒明显刺激大鼠CCSM细胞中cAMP水平升高[(48.7±1.1)nmol/L对(12.3±1.2)nmol/L和(7.8±1.4)nmol/L,P均<0.01],培养液中可检测到免疫反应性hCGRP。结论:可分泌表达生物活性肽重组腺相关病毒基因转移系统可有效进行多肽类在CCSM细胞过表达,并影响该细胞的cAMP水平,可被应用于ED的基因治疗。     

siRNA敲减整合素连接激酶影响人阴茎海绵体平滑肌细胞功能的体外研究

目的 探讨整合素连接激酶( ILK)基因对人海绵体平滑肌细胞收缩和舒张功能的影响.方法 人海绵体平滑肌细胞为实验对象,分为非沉默组、非处理对照组和ILK小干扰RNA(siRNA)实验组,采用siRNA干扰技术下调细胞中ILK的表达,伸展实验和基质胶黏附实验分别检测ILK基因敲减前后海绵体平滑肌细胞伸展和黏附能力变化,Transwell小室观察ILK表达对海绵体平滑肌细胞迁移能力的影响,荧光标记的鬼笔环肽染色海绵体平滑肌细胞观察ILK基因敲减前后细胞 骨架的变化.结果 黏附实验中,非沉默siRNA组、非处理对照组和实验组siRNA干扰的平滑肌细胞吸光度(A)值分别为0.184±0.034、0.179±0.028和0.092±0.010;迁移实验中,3组穿过Matrigel胶包被的Transwell小室的平滑肌细胞A值分别为0.184±0.017、0.188 ±0.013和0.106±0.003,实验组与非沉默siRNA组比较差异有统计学意义(P＜0.05);非处理对照组平滑肌细胞应力纤维错综分布于细胞周边与胞质内,而实验组的细胞应力纤维仅分布于细胞边缘.结论 ILK下调降低人海绵体平滑肌细胞的黏附、伸展、迁移能力,并通过影响细胞骨架重构参与调节平滑肌收缩与舒张状态,提示ILK基因有可能成为勃起功能障碍基因治疗的有效靶点.     

肿瘤细胞上清液对成纤维细胞表型转变及血管内皮生长因子-A表达的影响

目的检测肿瘤细胞上清液对成纤维细胞的激活情况及激活后血管内皮生长因子-A(VEGF-A)表达的变化规律。方法 MTT法检测普通培养液、含转化生长因子-β1(TGF-β1)的普通培养液以及肿瘤细胞上清液组成的条件培养液对成纤维细胞生长情况的影响,用RT-PCR、Western blot及免疫组织化学法检测不同培养条件下成纤维细胞的α-平滑肌肌动蛋白(α-SMA)与VEGF-A的表达。结果肿瘤细胞上清液对成纤维细胞的生长有一定的促进作用。含TGF-β1的普通培养液比普通培养液更有利于成纤维细胞转化为肌成纤维细胞,并且能维持肌成纤维细胞的表型,但是二者均不表达VEGF-A;条件培养液能促进成纤维细胞稳定表达α-SMA和VEGF-A,二者在培养后第1天均开始表达,第3天表达量达到峰值,第3天以后表达稳定。结论肿瘤细胞上清液能够有效、稳定地激活成纤维细胞为肌成纤维细胞,成纤维细胞的激活程度影响VEGF-A的表达,二者均具有最佳的激活时间点,并且最佳时间点相一致,最佳激活点的成纤维细胞有望成为一种可用于移植的促进血管重建的细胞。     

Observations on human smooth muscle cell cultures from hyperplastic lesions of prosthetic bypass grafts: production of a platelet-derived growth factor-like mitogen and expression of a gene for a platelet-derived growth factor receptor--a preliminary study

Prosthetic bypass grafts placed to the distal lower extremity often fail because of an occlusive tissue response in the perianastomotic region. The origin of the cells that comprise this occlusive lesion and the causes of the cellular proliferation are not known. To increase our understanding of this process we cultured cells from hyperplastic lesions obtained from patients at the time of reexploration for lower extremity graft failure, and we studied their identity and growth factor production in tissue culture. These cultures contain cells that express muscle-specific actin isoforms, shown by immunohistochemical staining, consistent with vascular smooth muscle origin. These cultures also released material that stimulated smooth muscle cell growth. A portion of this activity was similar to platelet-derived growth factor, since preincubation with antibody-to-human platelet-derived growth factor partially blocked the mitogenic effect of medium conditioned by human anastomotic hyperplastic cells. These conditioned media also contained material that competed with platelet-derived growth factor for its receptor, as measured in a radioreceptor assay. Northern blot analysis showed that these cells contain messenger RNA that encodes the A chain but not the B chain of platelet-derived growth factor. In addition, these cells contain messenger RNA that encodes a platelet-derived growth factor receptor. We conclude that cultured smooth muscle cells from human anastomotic hyperplastic lesions express genes for platelet-derived growth factor A chain and a platelet-derived growth factor receptor and secrete biologically active molecules similar to platelet-derived growth factor.(ABSTRACT TRUNCATED AT 250 WORDS)     

Migration of cells from human anterior cruciate ligament explants into collagen-glycosaminoglycan scaffolds.

Regeneration of the human anterior cruciate ligament after complete rupture offers several theoretical advantages over reconstruction, including maintenance of the complex insertion sites and fan-shape of the ligament and preservation of remaining proprioceptive fibers within the ligament substance. Well vascularized connective tissues, such as dermis, heal as a result of migration of fibroblasts into a provisional scaffold, the fibrin clot. Wound closure is subsequently facilitated by a contractile cell phenotype. This study was designed to determine if fibroblasts intrinsic to the human anterior cruciate ligament were capable of migrating from their native extracellular matrix onto an adjacent provisional scaffold in vitro. Another objective was to determine whether any of the cells that successfully migrated into the scaffold expressed the contractile actin isoform, alpha-smooth muscle actin, associated with wound contraction in other tissues. The results demonstrated that the cells intrinsic to the human anterior cruciate ligament were able to migrate into a collagen-glycosaminoglycan scaffold, bridging a gap between transected fascicles in vitro. As a result of this cell migration and proliferation, areas in the scaffold contained cell number densities similar to those seen in the human anterior cruciate ligament in vivo. No extracellular matrix or tissue formation was seen in the gap between directly apposed transected ends of the anterior cruciate ligament explants cultured without an interposed collagen-glycosaminoglycan scaffold. The fascicle-collagen-glycosaminoglycan-fascicle constructs and the fascicle-fascicle explants displayed minimal adherence after 6 weeks in culture. Any disruption in the contact area between explant and scaffold, even as small a gap as 50 microm, prevented cell migration from the explant to the collagen-glycosaminoglycan scaffold at the area of loss of contact. All cells that migrated into the scaffold at early time periods expressed the alpha-smooth muscle actin isoform. These results demonstrate that cells that migrate into and proliferate within the collagen-glycosaminoglycan matrix have contractile potential as reflected in their expression of the alpha-smooth muscle actin isoform. The role of these contractile cells in the healing process warrants further investigation. Moreover, this study demonstrates the potential of cells intrinsic to the human anterior cruciate ligament to migrate into collagen-glycosaminoglycan scaffolds that may ultimately be investigated as implants to facilitate ligament healing and regeneration.     

大鼠颈静脉分支-颈动脉间置模型新生内膜细胞来源研究

目的探讨大鼠颈静脉分支-颈动脉间置模型新生内膜细胞来源。方法建立SD大鼠颈静脉分支-颈动脉间置模型,分别于术后1、3、7、14和28d取静脉移植血管,定量分析新生内膜厚度,并进行α-SM—actin和CD34免疫组织化学分析。结果新生内膜增生在28d时最明显,血管吻合部位狭窄最严重,增生厚度近端（65．2±4．6）μm,远端（64．7±5．3）μm,中段（63．5±5．6）μm。新生内膜细胞主要来源于内皮细胞、相邻动脉血管平滑肌细胞或循环祖细胞,以新生内膜腔面为著。结论静脉移植血管新生内膜细胞主要来源于静脉移植血管本身内皮细胞、相邻动脉血管平滑肌细胞或循环祖细胞,提示可于血管吻合完成后进行局部干预或术后尽早全身用药,以防止移植血管狭窄。     

Expression of smooth muscle actin in cells involved in distraction osteogenesis in a rat model.

Distraction osteogenesis has proven to be of great value for the treatment of a variety of musculoskeletal problems. Little is still known, however, about the phenotypic changes in the cells participating in the bone formation process, induced by the procedure. Recent findings of the expression of a contractile muscle actin isoform, alpha-smooth muscle actin (SMA), in musculoskeletal connective tissue cells prompted this immunohistochemical study of the expression of SMA in cells participating in distraction osteogenesis in a rat model. The tissues within and adjacent to the distraction site could be distinguished histologically on the basis of cell morphology, density, and extracellular matrix make-up. The percentage of SMA-containing cells within each tissue zone was graded from 0 to 4. The majority of the cells in each of the zones stained positive for SMA within five days of the distraction period. The SMA-containing cells included those with elongated morphology in the center of the distraction site and the active osteoblasts on the surfaces of the newly forming bone.These finding warrant further investigation of the role of this contractile actin isoform in distraction osteogenesis and investigation of the effects of modulation of this actin isoform on the procedure.     

Decreased trabecular smooth muscle and caveolin-1 expression in the penile tissue of aged rats.

PURPOSE: Because decreased trabecular smooth muscle content is reportedly associated with vasculogenic impotence in men, we performed a rodent study to investigate the effect of aging on trabecular smooth muscle content and caveolin-1 protein expression in penile smooth muscle cells. MATERIALS AND METHODS: In 6 young (age 3 months) and 6 old (age 24 months) rats erectile function was evaluated by cavernous nerve stimulation. At sacrifice penile tissue samples were collected for Western blot analysis, Masson's trichrome staining, caveolin-1 immunostaining and electron microscopy. The percent of smooth muscle in the trabecular tissue was assessed by computer assisted image analysis. RESULTS: In the aged rats mean intracavernous pressure plus or minus standard deviation was decreased (70 +/- 8.8 versus 107 +/- 12.3 cm. water) and the latency period was increased (7.8 +/- 1.2 versus 4.5 +/- 0.5 seconds) significantly compared to values in the young rats (p <0.001). The mean ratio of trabecular smooth muscle-to-connective tissue was also significantly altered in old versus young rats (27% +/- 2.9% versus 42.1% +/- 5.1%, p <0.001). Immunostaining for caveolin-1 was noted in each group in the sarcolemma of smooth muscle cells and endothelium of trabecular sinusoids but the staining pattern was less intense and the percent of smooth muscle positive for caveolin-1 was decreased in aged versus young rats (17.9% +/- 2.5% versus 27.5% +/- 3.6%, p <0.001). Moreover, young trabecular smooth muscle cells had more caveolae in the sarcolemma on electron microscopy and a higher expression of caveolin-1 protein on Western blot analysis. In contrast, higher endothelial nitric oxide synthase protein expression was noted in the penile tissue of old rats. CONCLUSIONS: In these aged rats the decreased ratio of trabecular smooth muscle-to-collagen and the reduced expression of caveolin-1 may contribute to erectile dysfunction.