Heme oxygenase 1 (HO-1) challenges the angiogenic switch in prostate cancer.

Prostate cancer (PCa) is the second leading cause of cancer-associated death in men. Once a tumor is established it may attain further characteristics via mutations or hypoxia, which stimulate new blood vessels. Angiogenesis is a hallmark in the pathogenesis of cancer and inflammatory diseases that may predispose to cancer. Heme oxygenase-1 (HO-1) counteracts oxidative and inflammatory damage and was previously reported to play a key role in prostate carcinogenesis. To gain insight into the anti-tumoral properties of HO-1, we investigated its capability to modulate PCa associated-angiogenesis. In the present study, we identified in PC3 cells a set of inflammatory and pro-angiogenic genes down-regulated in response to HO-1 overexpression, in particular VEGFA, VEGFC, HIF1α and α5ß1 integrin. Our results indicated that HO-1 counteracts oxidative imbalance reducing ROS levels. An in vivo angiogenic assay showed that intradermal inoculation of PC3 cells stable transfected with HO-1 (PC3HO-1) generated tumours less vascularised than controls, with decreased microvessel density and reduced CD34 and MMP9 positive staining. Interestingly, longer term grown PC3HO-1 xenografts displayed reduced neovascularization with the subsequent down-regulation of VEGFR2 expression. Additionally, HO-1 repressed nuclear factor κB (NF-κB)-mediated transcription from an NF-κB responsive luciferase reporter construct, which strongly suggests that HO-1 may regulate angiogenesis through this pathway. Taken together, these data supports a key role of HO-1 as a modulator of the angiogenic switch in prostate carcinogenesis ascertaining it as a logical target for intervention therapy.

Hepatic damage and oxidative stress induced by Griseofulvin in mice.

Erythropoietic Protoporphyria (EPP) is a disease associated with ferrochelatase deficiency, which produces accumulation of protoporphyrin IX (PROTO IX) in erythrocytes, liver and skin. In some cases, a severe hepatic failure and cholestasis was observed. Griseofulvin (Gris) develops an experimental EPP with hepatic manifestations in animals. The aim of this work was to further characterize this model studying its effect on different metabolisms in mice Gris feeding (0-2.5%, 7 and 14 days). PROTO IX accumulation in liver, blood and feces, induction of ALA-S activity, and a low rate of Holo/Apo tryptophan pyrrolase activity was produced, indicating a reduction of free heme pool. The progressive liver injury was reflected by the aspect and the enlargement of liver and the induction of hepatic damage. Liver redox balance was altered due to porphyrin high concentrations; as a consequence, the antioxidant defense system was disrupted. Heme oxygenase was also induced, however, at higher concentrations of antifungal, the free heme pool would be so depleted that this enzyme would not be necessary. In conclusion, our model of Protoporphyria produced liver alterations similar to those found in EPP patients.

Renal damage and death in weaned mice after oral infection with Shiga toxin 2-producing Escherichia coli strains.

Enterohaemorrhagic Escherichia coli (EHEC) O157:H7 infections are considered a public health problem in both developed and developing countries because of their increasing incidence and the severity of clinical presentation. Approximately 10% of infected patients develop complications such as haemolytic uraemic syndrome (HUS) characterized by acute renal failure, thrombocytopenia and haemolytic anaemia. The precise sequence of events leading to HUS is still understood incompletely. Because of the lack of a reproducible small animal model for EHEC infections, in vivo studies examining EHEC-host early interactions are limited and insufficient. The aim of this study was to characterize the weaned BALB/c mouse as a model of E. coli O157:H7 infection. In this paper we report that human Shiga toxin 2 (Stx2)-producing EHEC strains can adhere to the intestinal epithelium of weaned BALB/c mice, and produce local damage which leads to systemic disease and death in a percentage of infected mice. The lethality of the EHEC strain is closely age-dependent, and is related to the bacterial ability to colonize intestine and to produce Stx2. It can be concluded that the weaned BALB/c mouse can be used as a small animal model to study host early responses, and the role of bacterial pathogenic factors in the induction of systemic disease, thus providing a useful tool for the evaluation of therapeutic or vaccine approaches.

Nuclear translocation of haeme oxygenase-1 is associated to prostate cancer.

The role of oxidative stress in prostate cancer has been increasingly recognised. Acute and chronic inflammations generate reactive oxygen species that result in damage to cellular structures. Haeme oxygenase-1 (HO-1) has cytoprotective effects against oxidative damage. We hypothesise that modulation of HO-1 expression may be involved in the process of prostate carcinogenesis and prostate cancer progression. We thus studied HO-1 expression and localisation in 85 samples of organ-confined primary prostate cancer obtained via radical prostatectomy (Gleason grades 4-9) and in 39 specimens of benign prostatic hyperplasia (BPH). We assessed HO-1 expression by immunohistochemical staining. No significant difference was observed in the cytoplasmic positive reactivity among tumours (84%), non-neoplastic surrounding parenchyma (89%), or BPH samples (87%) (P=0.53). Haeme oxygenase-1 immunostaining was detected in the nuclei of prostate cancer cells in 55 of 85 (65%) patients but less often in non-neoplastic surrounding parenchyma (30 of 85, 35%) or in BPH (9 of 39, 23%) (P<0.0001). Immunocytochemical and western blot analysis showed HO-1 only in the cytoplasmic compartment of PC3 and LNCaP prostate cancer cell lines. Treatment with hemin, a well-known specific inducer of HO-1, led to clear nuclear localisation of HO-1 in both cell lines and highly induced HO-1 expression in both cellular compartments. These findings have demonstrated, for the first time, that HO-1 expression and nuclear localisation can define a new subgroup of prostate cancer primary tumours and that the modulation of HO-1 expression and its nuclear translocation could represent new avenues for therapy.

Nitric oxide mediates prostaglandins' deleterious effect on lipopolysaccharide-triggered murine fetal resorption.

Genital tract bacterial infections could induce abortion and are some of the most common complications of pregnancy; however, the mechanisms remain unclear. We investigated the role of prostaglandins (PGs) in the mechanism of bacterial lipopolysaccharide (LPS)-induced pregnancy loss in a mouse model, and we hypothesized that PGs might play a central role in this action. LPS increased PG production in the uterus and decidua from early pregnant mice and stimulated cyclooxygenase (COX)-II mRNA and protein expression in the decidua but not in the uterus. We also observed that COX inhibitors prevented embryonic resorption (ER). To study the possible interaction between nitric oxide (NO) and PGs, we administered aminoguanidine, an inducible NO synthase inhibitor. NO inhibited basal PGE and PGF(2alpha) production in the decidua but activated their uterine synthesis and COX-II mRNA expression under septic conditions. A NO donor (S-nitroso-N-acetylpenicillamine) produced 100% ER and increased PG levels in the uterus and decidua. LPS-stimulated protein nitration was higher in the uterus than in the decidua. Quercetin, a peroxynitrite scavenger, did not reverse LPS-induced ER. Our results suggest that in a model of septic abortion characterized by increased PG levels, NO might nitrate and thus inhibit COX catalytic activity. ER prevention by COX inhibitors adds a possible clinical application to early pregnancy complications due to infections.

Relevance of neutrophils in the murine model of haemolytic uraemic syndrome: mechanisms involved in Shiga toxin type 2-induced neutrophilia.

It has been demonstrated that infections due to Shiga toxins (Stx) producing Escherichia coli are the main cause of the haemolytic uraemic syndrome (HUS). However, the contribution of the inflammatory response in the pathogenesis of the disease has also been well established. Neutrophils (PMN) represent a central component of inflammation during infections, and patients with high peripheral PMN counts at presentation have a poor prognosis. The mouse model of HUS, by intravenous injection of pure Stx type 2 (Stx2), reproduces human neutrophilia and allows the study of early events in the course of Stx2-induced pathophysiological mechanisms. The aim of this study was to address the contribution of PMN on Stx2 toxicity in a murine model of HUS, by evaluating the survival and renal damage in mice in which the granulocytic population was depleted. We found that the absence of PMN reduced Stx2-induced lethal effects and renal damage. We also investigated the mechanisms underlying Stx2-induced neutrophilia, studying the influence of Stx2 on myelopoyesis, on the emergence of cells from the bone marrow and on the in vivo migration into tissues. Stx2 administration led to an accelerated release of bone marrow cells, which egress at an earlier stage of maturation, together with an increase in the proliferation of myeloid progenitors. Moreover, Stx2-treated mice exhibited a lower migratory capacity to a local inflammatory site. In conclusion, PMN are essential in the pathogenesis of HUS and neutrophilia is not merely an epiphenomenon, but contributes to Stx2-damaging mechanism by potentiating Stx2 toxicity.

Defective signalling in salivary glands precedes the autoimmune response in the non-obese diabetic mouse model of sialadenitis.

The spontaneous non-obese diabetic (NOD) mouse model of Sjögren's syndrome provides a valuable tool to study the onset and progression of both the autoimmune response and secretory dysfunction. Our purpose was to analyse the temporal decline of salivary secretion in NOD mice in relation to the autoimmune response and alterations in various signalling pathways involved in saliva secretion within each salivary gland. A progressive loss of nitric oxide synthase activity in submandibular and parotid glands started at 12 weeks of age and paralleled the decline in salivary secretion. This defect was associated with a lower response to vasoactive intestinal peptide in salivary flow rate, cAMP and nitric oxide/cGMP production. No signs of mononuclear infiltrates or local cytokine production were detectable in salivary glands in the time period studied (10-16 weeks of age). Our data support a disease model for sialadenitis in NOD mice in which the early stages are characterized by defective neurotransmitter-mediated signalling in major salivary glands that precedes the autoimmune response.

Epidermal growth factor prevents prepartum luteolysis in the rat.

We have previously reported that intrauterine (i/u) administration of epidermal growth factor (EGF 500 ng) on day (d) 21 of pregnancy delayed 19.0 +/- 0.6 h the onset of labor. Progesterone (P) is secreted by ovarian corpora lutea (CL) throughout gestation in the rat. Prepartum CL regression due to increased uterine cyclooxygenase I and prostaglandin F(2alpha) results in P withdrawal followed by labor. The aims of the present work were (i) to study whether EGF delayed-onset of labor was mediated by a mechanism that prevented CL regression; (ii) to determine amniotic fluid (AF) EGF in pregnant rats. Rats on d21 of pregnancy received i/u EGF (500 ng) and were killed 0, 4, 8, 12, 24, and 48 h later. Control AF from rats on d13 and 18-22 of pregnancy was obtained. EGF decreased uterine prostaglandin F(2alpha) synthesis 8 h after treatment. Twelve hours after EGF injection, P reached its highest serum level and uterine cyclooxygenase I expression was undetectable. CL from rats killed 8 and 12 h after EGF were similar to those from rats on d13 of pregnancy, when serum P is maximum. EGF in AF increased throughout gestation, reached a maximum on d21, and decreased before the onset of labor. We suggest that the effect of EGF on the onset of labor was mediated by an early effect on the uterus that prevented prepartum CL regression.

Non-linear material models for tracheal smooth muscle tissue.

The aim of this study is to investigate the hyperelastic material models to describe the non-linear stress-strain behavior of tracheal smooth muscle tissue. Specifically, the goal is to validate the material model with experimental data using different finite element models and discuss the trends in stress-strain behavior of smooth muscle tissue. Both 2D and 3D finite element analyses were carried out to estimate the stress-strain behavior of the smooth muscle tissue. The results obtained indicate that the developed Ogden material model is valid and useful in explaining the stress strain behavior of tracheal smooth muscle tissue under different conditions. Finite element simulation results of the stress-strain behavior in the transverse direction are presented.

Anisotropic properties of tracheal smooth muscle tissue.

The anisotropic (directional-dependent) properties of contracting tracheal smooth muscle tissue are estimated from a computational model based on the experimental data of length-dependent stiffness. The area changes are obtained at different muscle lengths from experiments in which stimulated muscle undergoes unrestricted shortening. Then, through an interative process, the anisotropic properties are estimated by matching the area changes obtained from the finite element analysis to those derived from the experiments. The results obtained indicate that the anisotropy ratio (longitudinal stiffness to transverse stiffness) is about 4 when the smooth muscle undergoes 70% strain shortening, indicating that the transverse stiffness reduces as the longitudinal stiffness increases. It was found through a sensitivity analysis from the simulation model that the longitudinal stiffness and the in-plane shear modulus are not very sensitive as compared to major Poisson's ratio to the area changes of the muscle tissue.

Molecular analysis of p53 tumor-suppressor gene and microsatellites in preneoplastic and neoplastic lesions of the colon and esophagus.

Mutations in exons 4-8 of the p53 gene by the PCR-SSCP analysis in preneoplastic and neoplastic lesions of the colon (n=11) and esophagus (n=18) were screened. p53 overexpression by immunohistochemistry in 11 colonic lesions and 13 microsatellites, in all the patients (n=29), were also studied. A positive result concordancy between the three techniques was found in 1 adenoma and 2 adenocarcinomas of the colon, each with loss of heterozygocity of microsatellites. Metaplastic lesions of esophagus showed biallelic mutations and low frequency of microsatellite alterations. The relationship between genetic alterations in p53, microsatellites and type of colon and esophageal lesions is discussed.

The time of tumor cell division and death depends on the site of growth.

We show here, for the first time, in two very different murine tumors, a mammary one (ectoderm) and a lung one (endoderm), that: tumors have day/night differences of spontaneous apoptosis additional to the well-known circadian rhythm of mitosis. The times of maximal and minimal mitosis and apoptosis changed for a tumor cell line when growing in different organs (as metastasis) or anatomical sites. Both tumor lines, have identical circadian curves when growing in a specific organ or anatomical site. The peaks of apoptosis match with the valleys of mitosis and vice versa.

H(2)O(2) mediates Ca(2+)- and MLC(20) phosphorylation-independent contraction in intact and permeabilized vascular muscle.

One purpose of the current study was to establish whether vasoconstriction occurs in all vessel types in response to H(2)O(2). Isometric force was measured in pulmonary venous and arterial rings, and isobaric contractions were measured in mesenteric arteries and veins in response to H(2)O(2). A second purpose was to determine whether H(2)O(2)-induced contraction is calcium independent. The addition of H(2)O(2) to calcium-depleted (using the Ca(2+) ionophore ionomycin in zero calcium EGTA buffer) muscle caused contraction. Furthermore, permeabilized muscle contracted in response to H(2)O(2) even in zero Ca(2+). The final purpose was to determine whether the 20-kDa regulatory myosin light chain (MLC(20)) phosphorylation plays a role in H(2)O(2)-induced contraction. Pulmonary arterial strips were freeze-clamped at various time points during H(2)O(2)-induced contractions, and the relative amounts of phosphorylated MLC(20) were measured. H(2)O(2) caused dose-dependent contractions that were independent of MLC(20) phosphorylation. ML-9, a myosin light chain kinase inhibitor, had no effect on the H(2)O(2) contractile response. In conclusion, H(2)O(2) induces Ca(2+)- and MLC(20) phosphorylation-independent contraction in pulmonary and systemic arterial and venous smooth muscle.

Deferoxamine reduces tissue injury and lethality in LPS-treated mice.

We studied the effect of deferoxamine (DFX), an iron chelator, which can also act as a free radical scavenger, in an experimental murine model of sepsis. In vivo studies demonstrated that pretreatment of mice with DFX reduces tumor necrosis factor alpha (TNF-alpha) serum levels and increases the rate of survival of mice inoculated with lethal doses of lipopolysaccharide (LPS) or Escherichia coli O111:B4. By using the iron chelated form of DFX (ferrioxamine) the same results were obtained, suggesting that in this model, DFX could act as a free radical scavenger. On the other hand, DFX prevents mortality induced either by LPS or murine recombinant TNF-alpha in D(+)-galactosamine (GalN)-sensitized mice. These protective actions of DFX correlate with an attenuated tissue damage observed in lungs, livers and kidneys of LPS-treated animals and GalN-sensitized mice inoculated with TNF-alpha.

Terapia fotodinámica en el tratamiento de tumores de piel inducidos quimicamente / Photodynamic therapy in the skin tumors tratment chemical induced

La terapia fotodinámica (TFD) es un tratamiento para tumores que utiliza luz y compuestos fotosensibles como porfirinas endógenas sintetizadas a partir del precursor ácido 5-Aminolevúlico (ALA). En el presente trabajo se estudió la acumulación de porfirinas luego de la aplicación de ALA tópico en diferentes vehículos y el grado de efectividad del tratamiento fotodinámico en tumores cutáneos inducidos químicamente en ratones SENCAR. Cada tipo de tumor (papilomas, queratoacantomas y carcinomas epidermoides) presentó una respuesta distinta al ALA-TFD. Sobre los carcinomas in situ e invasores, la acción de la TFD fue más evidente, observándose degeneración y desestructuración de los estratos más superficiales, hasta 1,5 mm, con cambios destructivos asociados a coagulación. Sobre la vascularización de la dermis se vieron fenómenos de congestión y hemorragia. Los efectos más encontrados fueron vacuolización y condensación de citoplasma, picnosis y desintegración nuclear. La acción sobre los papilomas en cambio, fue más limitada, debido a la escasa penetración del ALA y o la luz a través de la extensa capa de queratina. Sólo se encontraron respuestas talkes como concurrencia de mastocitos, infiltrados de leucocitos y esclerohialinizacion. Tambien se estudió la acumulación de porfirinas en los papilomas luego de la topicación de ALS en distintos vehículos sobre la superficie tumoral. La aplicación del ALA en loción fue la que mayor síntesis de porfirinas indujo (loción salina=2,31 mas menos 0,52 mi g/g. Con la aplicaciuón de una emulsión de aceite en agua, la acumulación de porfirinas fue de 1,30 mas menos 0,23 mi g/g y con una emulsión de agua en aceite, la concentración fue de 0,62 mas menos 0,12 mi g/g. Se concluye, que la TFD a partir de ALA es indicada para lesiones cutáneas con escasa queratinización, en particular para lesiones superficiales. El ALA topicado en loción sola o con etanol son los vejículos más efectivos para tratar estas lesiones

Terapia fotodinámica en el tratamiento de tumores de piel inducidos quimicamente / Photodynamic therapy in the skin tumors tratment chemical induced

La terapia fotodinámica (TFD) es un tratamiento para tumores que utiliza luz y compuestos fotosensibles como porfirinas endógenas sintetizadas a partir del precursor ácido 5-Aminolevúlico (ALA). En el presente trabajo se estudió la acumulación de porfirinas luego de la aplicación de ALA tópico en diferentes vehículos y el grado de efectividad del tratamiento fotodinámico en tumores cutáneos inducidos químicamente en ratones SENCAR. Cada tipo de tumor (papilomas, queratoacantomas y carcinomas epidermoides) presentó una respuesta distinta al ALA-TFD. Sobre los carcinomas in situ e invasores, la acción de la TFD fue más evidente, observándose degeneración y desestructuración de los estratos más superficiales, hasta 1,5 mm, con cambios destructivos asociados a coagulación. Sobre la vascularización de la dermis se vieron fenómenos de congestión y hemorragia. Los efectos más encontrados fueron vacuolización y condensación de citoplasma, picnosis y desintegración nuclear. La acción sobre los papilomas en cambio, fue más limitada, debido a la escasa penetración del ALA y o la luz a través de la extensa capa de queratina. Sólo se encontraron respuestas talkes como concurrencia de mastocitos, infiltrados de leucocitos y esclerohialinizacion. Tambien se estudió la acumulación de porfirinas en los papilomas luego de la topicación de ALS en distintos vehículos sobre la superficie tumoral. La aplicación del ALA en loción fue la que mayor síntesis de porfirinas indujo (loción salina=2,31 mas menos 0,52 mi g/g. Con la aplicaciuón de una emulsión de aceite en agua, la acumulación de porfirinas fue de 1,30 mas menos 0,23 mi g/g y con una emulsión de agua en aceite, la concentración fue de 0,62 mas menos 0,12 mi g/g. Se concluye, que la TFD a partir de ALA es indicada para lesiones cutáneas con escasa queratinización, en particular para lesiones superficiales. El ALA topicado en loción sola o con etanol son los vejículos más efectivos para tratar estas lesiones(AU)

Two different types of concomitant resistance induced by murine tumors: morphological aspects and intrinsic mechanisms.

Concomitant resistance (CR) is the phenomenon according to which a tumor-bearing host inhibits the growth of a secondary implant of the same tumor at a distant site. Confirming and extending previous results of our laboratory, histological studies have revealed that two temporally separate peaks of CR can be detected throughout tumor evolution. The first peak induced by immunogenic small tumors, in euthymic but not in nude mice, is associated with extensive necrosis of the secondary tumor implant and a profuse infiltration of polymorphonuclear granulocytes and mononuclear cells resulting in its final destruction; these features correspond to a typical immunological rejection. The second peak of CR induced by both immunogenic and non-immunogenic large tumors, in euthymic as well as in nude mice, is characterized by a dormant tumor stage with scarce or null mononuclear infiltration, associated with a significant reduction of tumor mitotic index and of the number of PCNA+ cells along with an increase in apoptosis and an arrest in S phase. In previous reports we suggested that a 1000 D serum fraction from mice bearing large tumors could be responsible for the induction of this dormant tumor stage. In this study tumor cells incubated in vitro with that serum factor mimicked the inhibition and cellular alterations observed in vivo in the secondary tumor inhibited by the second peak of CR. Moreover, the passive transfer of this factor by the intra-peritoneal (i.p.) route induced an in vivo inhibition of an i.p. tumor reproducing the image characteristic of the second peak of CR. This represents a direct proof that this serum factor can restrain tumor growth in vivo and that it is, most probably, the effector of the second peak of CR.

Topical and intratumoral photodynamic therapy with 5-aminolevulinic acid in a subcutaneous murine mammary adenocarcinoma.

One of the most promising substances used in photodynamic therapy (PDT) is 5-aminolevulinic acid (ALA), which induces endogenous synthesis and accumulation of porphyrins in malignant cells. In this paper we have shown that both topical and intratumoral administration of ALA in a subcutaneously implanted mammary carcinoma produced a significant synthesis of porphyrins and subsequent sensitization to laser light. Porphyrin accumulation was greater when ALA was administered intratumorally and tumour/normal skin porphyrin concentration ratios were higher compared with topical application. Irradiation was optimal between 2 and 3 h after topical application of 50 mg of a 20% ALA cream and 2-4 h after intratumoral administration of 30 mg ALA/cm3. The pattern of tumour response evaluated as the delay of tumour growth was similar following either route of drug administration. Applications of PDT were performed once, twice or three times in the study. The response to successive applications was constant for the same tumour, indicating that no resistance was acquired. Microscopic analysis showed both induction of foci of necrosis and haemorrhage, morphological features of apoptotic cells and total absence of cellular immune response. This paper reports on PDT with topical ALA in a subcutaneous carcinoma leading to tumour growth delay. These findings may have great relevance in the treatment of cutaneous metastasis of mammary carcinomas.

Inhibition of metastases by a serum factor associated to concomitant resistance induced by unrelated murine tumors.

Murine lung metatases growing undisturbed by the primary tumor were significantly inhibited by the concomitant resistance induced by a secondary subcutaneous implant of two unrelated tumors. Such inhibition was T-independent since it was also observed in nude mice; its full expression was dependent on the presence of the secondary tumor implant and it was exerted on both macroscopic and microscopic established metastases and not on the process of tumor cell dissemination from the primary tumor. Direct and indirect mechanisms seemed to be involved, the former affecting the metastatic cells per se by causing a decrease in proliferation and an increase in apoptosis while the latter affected neo-vascularization. These antitumor and antiangiogenic effects could be attributed to a serum factor induced by the unrelated tumors generating concomitant resistance. This factor proved to be heat, acid and alkaline resistant and dialysable; it was recovered in an HPLC column with maximum absorption at 215 and 266 nm; it was anionic at neutral pH, exhibiting free carboxil groups and one or more molecules of tyrosine, with a molecular weight between 870 and 1300 Dalton. Intravenous administration of this factor significantly inhibited lung metastases, decreasing mitosis and increasing apoptosis similar to that observed in the presence of the unrelated tumors.

Influence of intercellular tissue connections on airway muscle mechanics.

Contraction of smooth muscle in visceral organs is modified by structures external to the muscle. Within muscle tissue itself, connective tissue plays an important role in force transference among the contractile cells. Connections arranged radially can affect contractile mechanics by limiting tissue expansion at short lengths. Previous work suggests that increased stiffness at extreme shortening is due to such radial constraints. Two approaches to further study of these effects are reported. To increase radial constraints, very thin Silastic bands were placed loosely about strips of canine trachealis muscle at rest length. The strips were allowed to shorten under light afterloads, expanding until restrained by the bands. Subsequent removal of the bands allowed increased shortening, with less increase in stiffness at short lengths. Related isometric effects were observed. To reduce constraints, muscle strips were partially digested with collagenase. Compared with control conditions, this treatment permitted further shortening, with less increase in stiffness at short lengths. These results emphasize the role of extracellular structures in determining mechanical function of smooth muscle.