Systemic iron reduction via an iron deficient diet decreases the severity of knee cartilage lesions in the Dunkin-Hartley guinea pig model of osteoarthritis.

OBJECTIVE: Iron accumulation is emerging as a player in aging-related disorders due to its propensity for generating reactive oxygen species (ROS). Studies investigating the role of iron in the pathogenesis of primary osteoarthritis (OA) are limited. We designed a proof-of-principle study to determine the effect of systemic iron deficiency, via an iron deficient diet, on knee OA in an animal model. METHODS: Twelve-week-old male Hartley guinea pigs received the standard diet (n = 6) or a diet devoid of iron (n = 6) for 19-weeks. Iron levels were determined in the serum, liver, and articular cartilage. Knees were collected to assess structural changes related to OA (microcomputed tomography, histopathology). Immunohistochemistry was performed to evaluate the presence and distribution of a disintegrin and metalloproteinase with thrombospondin motifs 4 (ADAMTS4) and ROS-driven 4-hydroxynonenal (4-HNE)-induced protein adducts. Transcript expression was also assessed. RESULTS: Relative to control animals, an iron deficient diet reduced the concentration of this mineral in serum, liver, and articular cartilage. Iron deficient animals had lower histologic OA scores; decreased subchondral bone mineral density was also noted. This reduction in knee joint pathology was accompanied by a decrease in: ADAMTS4 in synovium; and 4-HNE protein adducts from lipid peroxidation in both the menisci and articular cartilage of iron deficient animals. Expression of iron-related genes in these tissues was also altered in treated animals. CONCLUSIONS: Results from this study suggest that systemic iron levels may play a role in knee OA pathogenesis, with a short-term deficit in dietary iron reducing the severity of knee cartilage lesions.

Clinical and Histologic Manifestations of a Novel Rectus Femoris Myotendinous Junction Injury in Rats.

Background: Animal models of muscle injury have primarily relied on methods which do not mimic the chronic scarring that typically occurs adjacent to the myotendinous junction (MTJ). The goal of this study was three-fold: (i) to create a strain-induced in vivo model of rectus femoris MTJ injury in rats; (ii) to document clinical manifestations of injury using longitudinal tracking of individual animals via voluntary and compulsory (treadmill) mobility analyses and (iii) to validate and assess the model for persistent scarring through serial histologic assessment and development of a semi-quantitative grading scheme to characterize injury response over time. Methods: Strain-induced MTJ injury was generated in male Sprague Dawley rats via needle tension directed along the transverse axis between the rectus femoris muscle and distal tendon that attaches to the patella. Animals received mobility assessments (gait analysis using a DigiGait Treadmill System and weight bearing using a Tekscan Rodent Walkway System) at days 0, 1, 3, 6, 13, 20, and 27 of the experimental protocol. Rats were euthanized at 1, 3, 7, 14, and 28 days post-injury (n = 6 rats per time-point) and hindlimbs were processed for histology. Results: Significant changes in locomotor parameters included injured and contralateral limb paw area, max dA/dt (limb deceleration/breaking time), stride time, stance time, force time impulse, and fore/hind symmetry, and injured limb maximum force. The most significant and consistent histologic finding was a pathologic fibrotic adhesive lesion at the muscle and tendon interface along the proximal aspect of the patella just distal to the injury site. This lesion was composed of reactive fibroblasts, disorganized collagen fibers, vascular profiles, and a myxomatous ground substance stroma. Conclusions: This work is the first to characterize the clinical and pathologic development of a chronic model of rectus femoris MTJ injury, which resulted in altered mobility likely caused by a strain-induced fibrotic scar along the anterior patella. Notably, both the functional and pathologic changes recapitulated the course of injury progression similar to what is described in humans. This work provides a unique model to study MTJ injury mechanisms for the identification of enhanced treatment options for patients who suffer from activity-related muscle conditions.

Systemic iron overload exacerbates osteoarthritis in the strain 13 guinea pig.

OBJECTIVE: Iron is emerging as a key player in aging-associated diseases due to its propensity for driving free radical formation. Studies examining the role of iron in the pathogenesis of primary osteoarthritis (OA) are limited. Our objective was to establish a direct relationship between excess iron and OA by administering iron dextran to a guinea pig strain with decreased propensity for developing this disease. DESIGN: Twenty, 12-week-old Strain 13 guinea pigs received either iron dextran or dextran control intraperitoneally once weekly for 4 weeks; termination occurred at 16 weeks of age. Iron levels were determined systemically (serum and liver) and within diarthrodial joints [femoral head articular cartilage and infrapatellar fat pads (IFPs) of knee joints]. One knee was collected to score structural changes associated with OA via microcomputed tomography (microCT) and histology using published grading schemes. Articular cartilage and IFPs were harvested from contralateral knees for gene expression analyses. RESULTS: Iron overload was confirmed systemically via increased serum iron and liver iron concentration. Articular cartilage and IFPs in the iron dextran group also had higher levels of iron. Excess iron worsened knee OA using both microCT and histologic scoring systems. Gene analyses revealed that exogenous iron altered the expression of iron trafficking proteins, select cytokines, and structural components of cartilage. CONCLUSION: These results demonstrate that systemic iron overload caused cellular iron accumulation in the knee joint. This excess iron is associated with increased expression of local inflammatory mediators and early onset and progression of knee joint OA in Strain 13 animals.

Quantitative Gait Analysis Detects Significant Differences in Movement between Osteoarthritic and Nonosteoarthritic Guinea Pig Strains before and after Treatment with Flunixin Meglumine.

A computer-aided gait analysis system was used to contrast two guinea pig strains with differing propensity for osteoarthritis (OA), with/without administration of a nonsteroidal anti-inflammatory drug. Walking speed and static/dynamic gait parameters were determined at baseline. Flunixin meglumine was given and animals were evaluated 4, 24, and 72 hours after treatment. Body weight was compared using unpaired t-tests. Knee joints were histologically evaluated using species-specific criteria; indices were analyzed using one-way ANOVA, Kruskal-Wallis test, followed by Dunn's multiple comparisons. A generalized linear model followed by Tukey's posttests juxtaposed gait parameters; walking speed was a covariate for other outcome measures. Body weight was not different between strains; OA-prone animals demonstrated more progressive chondropathy. At baseline, OA-prone animals had slower walking speeds, narrower hind limb bases of support, shorter stride lengths, and slower limb swing speeds relative to OA-resistant animals. These differences were not detected 4 or 24 hours after treatment. By 72 hours, OA-prone animals had returned to baseline values. These findings indicate a distinct voluntary gait pattern in a rodent model of bilateral primary OA, modification of which may allow rapid screening of novel therapies. Flunixin meglumine temporarily permitted OA-prone animals to move in a manner that was analogous to OA-resistant animals.

In vivo reduction or blockade of interleukin-1ß in primary osteoarthritis influences expression of mediators implicated in pathogenesis.

OBJECTIVE: Diminish interleukin-1ß (IL-1ß) signaling in a model of primary osteoarthritis by RNA interference-based transcript reduction or receptor blockade, and quantify changes incurred on transcript expression of additional mediators. METHODS: Knees of Hartley guinea pigs were collected at 120 and 180 days of age following injection with viral vectors (N = 4/treatment group/date) at 60 days. Two groups received either adeno-associated viral serotype 5 vector containing a knockdown sequence (TV), or adenoviral vector encoding for IL-1 receptor antagonist protein (Ad-IRAP); treatments were contrasted with opposite knees administered corresponding vector controls. A third group evaluated TV relative to saline-only injected knees. Chondropathy and immunohistochemistry findings were compared to untreated guinea pigs. Transcript expression levels in cartilage were calculated using the comparative CT (2(-ΔΔCT)) method and analyzed by one-way analysis of variance (ANOVA) with pairwise comparisons using Tukey 95% confidence intervals. RESULTS: Vector transduction was confirmed at both harvest dates. TV and Ad-IRAP, relative to vector controls, significantly decreased IL-1ß. Inflammatory mediators [tumor necrosis factor-α (TNF-α), IL-8, interferon-γ (IFN-γ)], and catabolic matrix metalloproteinase 13 (MMP13) were also decreased, while anabolic transforming growth factor-ß1 (TGF-ß1) was increased. IL-1ß was also decreased by TV vs saline, with a decrease in MMP13 and increase TGF-ß1; TNF-α, IL-8, and IFN-γ were transiently increased. CONCLUSIONS: This work confirmed that a reduction in IL-1ß signaling was accomplished by either method, resulting in decreased expression of three inflammatory mediators and one catabolic agent, and increased expression of an anabolic molecule. Thus, evidence is provided that IL-1ß serves a role in vivo in spontaneous osteoarthritis and that these translational tools may provide beneficial disease modification.

Effective reduction of the interleukin-1ß transcript in osteoarthritis-prone guinea pig chondrocytes via short hairpin RNA mediated RNA interference influences gene expression of mediators implicated in disease pathogenesis.

OBJECTIVE: To ascertain a viral vector-based short hairpin RNA (shRNA) capable of reducing the interleukin-1ß (IL-1ß) transcript in osteoarthritis (OA)-prone chondrocytes and detect corresponding changes in the expression patterns of several critical disease mediators. METHODS: Cultured chondrocytes from 2-month-old Hartley guinea pigs were screened for reduction of the IL-1ß transcript following plasmid-based delivery of U6-driven shRNA sequences. A successful plasmid/shRNA knockdown combination was identified and used to construct an adeno-associated virus serotype 5 (AAV5) vector for further evaluation. Relative real-time reverse transcription polymerase chain reaction (RT-PCR) was used to quantify in vitro transcript changes of IL-1ß and an additional nine genes following transduction with this targeting knockdown vector. To validate in vitro findings, this AAV5 vector was injected into one knee, while either an equivalent volume of saline vehicle (three animals) or non-targeting control vector (three animals) were injected into opposite knees. Fold differences and subsequent percent gene expression levels relative to control groups were calculated using the comparative CT (2(-ΔΔCT)) method. RESULTS: Statistically significant decreases in IL-1ß expression were achieved by the targeting knockdown vector relative to both the mock-transduced control and non-targeting vector control groups in vitro. Transcript levels of anabolic transforming growth factor-ß (TGF-ß) were significantly increased by use of this targeting knockdown vector. Transduction with this targeting AAV5 vector also significantly decreased the transcript levels of key inflammatory cytokines [tumor necrosis factor-α (TNF-α), IL-2, IL-8, and IL-12] and catabolic agents [matrix metalloproteinase (MMP)13, MMP2, interferon-γ (IFN-γ), and inducible nitrous oxide synthase (iNOS)] relative to both mock-transduced and non-targeting vector control groups. In vivo application of this targeting knockdown vector resulted in a >50% reduction (P=0.0045) or >90% (P=0.0001) of the IL-1ß transcript relative to vehicle-only or non-targeting vector control exposed cartilage, respectively. CONCLUSIONS: Successful reduction of the IL-1ß transcript was achieved via RNA interference (RNAi) techniques. Importantly, this alteration significantly influenced the transcript levels of several major players involved in OA pathogenesis in the direction of disease modification. Investigations to characterize additional gene expression changes influenced by targeting knockdown AAV5 vector-based diminution of the IL-1ß transcript in vivo are warranted.

Temporal expression and tissue distribution of interleukin-1ß in two strains of guinea pigs with varying propensity for spontaneous knee osteoarthritis.

OBJECTIVE: To provide a comprehensive immunohistochemical (IHC) map of the temporal expression and tissue distribution of interleukin-1ß (IL-1ß) through progression of osteoarthritis (OA) in two strains of guinea pigs with varying propensity for spontaneous knee joint disease. METHODS: OA-prone Hartley and OA-resistant Strain 13 guinea pigs were collected at 60, 120, 180, 240, 360, and 480 days of age (N=4 animals per strain per date). IHC was performed on whole joint preparations; the distribution of IL-1ß expression on coronal sections was mapped, semi-quantitatively scored, and correlated to OA grade using Mankin criteria with guinea pig-specific modifications. OA and IHC indices were compared among times and between strains using the Kruskal-Wallis one-way analysis of variance by ranks followed by Dunn's post test. RESULTS: OA indices for both strains increased from 60 to 480 days of age; a statistically higher score (P ≤ 0.01) was found in Hartley animals at 180, 240, 360, and 480 days. At 60 days of age, IL-1ß expression was detected in cartilage, menisci, synovium, and subchondral bone in both strains. Persistent and statistically increased (P<0.05) IL-1ß expression was found in these same tissues in Hartley animals at 120 and 180 days, while Strain 13 animals demonstrated a significant reduction in positive immunostaining. Statistical differences in IHC indices between strains beyond 240 days of age were restricted to synovium (days 240 and 480) and subchondral bone (days 360 and 480). CONCLUSIONS: As expected, histologic OA proceeded in an accelerated manner in Hartley animals relative to Strain 13 animals. The OA-prone strain did not demonstrate reduced IL-1ß expression during adult maturity as occurred in the OA-resistant strain, and this persistent expression may have corresponded to early incidence of OA. Future interventional studies are warranted to explore whether dysregulation of IL-1ß expression may contribute to premature onset of spontaneous disease in the Hartley guinea pig.

Aortic valve replacement: comparison of late survival between autografts and homografts.

BACKGROUND: Autografts (AG) and homografts (HG) are currently considered the best choices for replacement of the diseased aortic valve in young adults, although few data exist comparing their late outcome. Nonhomogeneous populations and evolving operative techniques confound existing comparisons. METHODS: To help clarify these issues, we reviewed our results with 238 hospital survivors (aged 17 to 82 years) undergoing operation between 1986 and 1999. All operations were done as root replacements, and patients needing concomitant valve replacement were excluded. RESULTS: Mean age of the 145 AGs and 93 HGs was 35 +/- 13 years and 49 +/- 17 years, respectively (p < 0.001). Previous aortic valve replacement was done in 12 (8%) AG and 32 (34%) HG patients (p = 0.001), and active endocarditis was present at time of current operation in 10 (7%) AG and 25 (27%) HG patients (p = 0.001). Maximum follow-up was 12.2 years for AGs and 12.8 years for HGs. Late survival at 10 years was 77% +/- 11% for AGs and 67% +/- 9% for HGs (p = 0.13). Freedom from AG or HG degeneration at 10 years was 97% +/- 2% and 79% +/- 10% (p = 0.63). Freedom from valve-related complications at 10 years was 73% +/- 10% and 64% +/- 10% (p = 0.93), respectively. Freedom from all reoperations at 10 years was 88% +/- 5% for AG and 72% +/- 11% for HG (p = 0.67). CONCLUSIONS: Autografts and HGs have comparable late survival. The incidence of valve degeneration is low for both AG and HG up to about 8 years at which point there may be a trend toward an advantage for AG over the HG, suggesting benefit for the younger patient.

Normalization of left ventricular dimensions after Ross operation with aortic annular reduction.

BACKGROUND: Fifty-seven patients (August 1995 to November 1998) with a dysplastic dilated aortic root, a relative contraindication to the Ross operation, received an extended Ross operation with aortic annulus reduction and external cuff fixation (age 14-54 years). To assess the efficacy of these operations, echocardiographic assessment of autograft valve function and left ventricular function and dimensions were reviewed. METHODS: Preoperative and postoperative assessment of 27 patients with aortic insufficiency (AI group) and 30 patients with aortic stenosis (>20 mm Hg peak gradient) and aortic insufficiency (AS group) were compared. Aortic annulus size, valvular gradient, valve insufficiency, left ventricular dimensions at end-systole and end-diastole, left ventricular fractional shortening, and left ventricular mass were assessed. RESULTS: There was one late death. Aortic annulus size, degree of AI, left ventricular internal dimensions, and left ventricular mass were all significantly reduced (p<0.05) postoperatively in the AI group. Mean peak pressure gradients for this group were 6.8+/-6.7 mm Hg before operation and 8.7+/-6.4 mm Hg at 1 year after operation. Peak pressure gradient, aortic annulus size, degree of AI, left ventricular internal dimensions, and left ventricular mass were significantly reduced (p<0.05) in the AS group. Mean fractional shortening was within normal limits pre- and postoperatively for both groups. CONCLUSIONS: Regression of left ventricular dilatation and hypertrophy, excellent autograft valve function, and survival suggest that this modification of the Ross operation may be offered to patients with a dysplastic aortic root requiring aortic valve replacement.

Advantage of autograft and homograft valve replacement for complex aortic valve endocarditis.

BACKGROUND: There are advantages to using homografts and autografts as aortic valve replacements, particularly in patients with infective endocarditis. To better define these advantages, we reviewed our 13-year experience with the surgical management of infective endocarditis involving the aortic valve and root. METHODS: From 1986 through 1998, 81 adults with aortic valve endocarditis underwent valve replacement (AVR). The mean age of the 65 men and 16 women was 44 +/- 14 years. Sixty-three (78%) patients had active endocarditis at the time of operation. Non-native valve endocarditis was present in 29 (36%) patients, in 9 of whom the infection was a recurrence. Aortic valve replacements were performed with 46 homografts (homo-AVR), 25 autografts (Ross-AVR), and 10 prosthetic valves (prosth-AVR). Among Ross-AVR and homo-AVR patients, 11 required mitral valve replacement or repair (homo-Ross DVR). Follow-up was 90% complete within 2 years of the end of the study with a mean of 3.7 +/- 3.4 years. RESULTS: Early mortality was 16% (13 of 81 patients). This was 12% (3 of 25 patients) for Ross-AVR, 17% (8 of 46 patients) for homo-AVR, and 20% (2 of 10 patients) for prosth-AVR. Overall late mortality was 10% (7 of 68 patients) with a valve-related late mortality of 7% (5 of 68 patients). Actuarial survival at 5 years was 88% +/- 9% in Ross-AVR, 69% +/- 11% in homo-AVR, and 29% +/- 22% in prosth-AVR (p = 0.03). Endocarditis recurred in 12.5% (1 of 8 patients) with prosth-AVR and 3% (2 of 60 patients) in homo-Ross AVR. CONCLUSIONS: Valve replacement in the presence of native and prosthetic endocarditis remains a formidable challenge. Autografts and homografts are the preferred replacement aortic valves for these patients even if concomitant mitral valve replacement is required, and risk of valve-related death or recurrent endocarditis is low at medium-term follow-up.

Pulmonary autograft replacement in children. The ideal solution?

Fifty-one children, aged 1.8 to 21 years (mean, 11.4) with aortic valve replacement using a pulmonary autograft are reviewed. Twenty-nine were intra-aortic implants and 22 were root replacements. There was one operative death, no late deaths, and two have required reoperation. Actuarial freedom from reoperation was 93% +/- 5.5 at 5.6 years. Freedom from progression of aortic insufficiency (AI) was 81% +/- 9 at 5.6 years in the intra-aortic implants and 86% +/- 10 in the root replacement. Enlargement of the pulmonary autograft was seen echocardiographically in both groups. This enlargement was consistent with somatic growth and not associated with progression of AI. Ten of 19 patients with aortic stenosis had an LV mass index suggestive of LV hypertrophy before operation. At 1 year, 18 of 25 had a normal LV mass index. Thirteen of 16 patients with AI had preoperative abnormal LV mass index. All but four returned to normal by 1 year. Low operative risk, excellent function, resolution of abnormal LV hemodynamics, and enlargement consistent with somatic growth suggest that the pulmonary autograft is the ideal replacement for the malfunctioning aortic valve.

Pulmonary autograft replacement of the aortic valve: an evolution of technique.

The use of the patient's pulmonary valve for replacement of the patient's diseased aortic valve was introduced and developed by Mr. Donald Ross. Its demonstrated durability, freedom from thromboembolism, and potential for growth has led to increased utilization of this technique. Modifications of the earlier techniques have led to a reproducible operation with low operative risk and excellent mid-term results.

Normal left ventricular function following pulmonary autograft replacement of the aortic valve in children.

To assess growth potential and hemodynamic sequelae of pulmonary autograft valves implanted into aortic outflow tracts of children, we reviewed our experience with 37 patients (2-21 years) from August 1986 to December 1990. Twenty patients had predominantly aortic stenosis (AS), and 17 had aortic insufficiency (AI). Operative mortality was 3%. Two technical failures required reoperation. Of survivors, six (18%) have moderate AI. Pre- and postoperative echocardiograms were reviewed. The AS group showed increased left ventricular (LV) cavity size by greater than 1-year follow-up, and decreased LV wall and interventricular septal thickness. In the AI group, wall and septal thickness increased by 10 days and LV cavity decreased by 10 days, 60 days, and greater than 1 year. Root replacements (n = 14) showed mean increases of 4.3 mm and 5.3 mm, respectively, in diameters of the aortic annulus and aortic sinuses at greater than 1 year. Intraaortic implants increased 3.1 mm (annulus) and 3.9 mm (sinuses) at greater than 1 year. The pulmonary autograft procedure is safe, and successful implantation normalizes LV dimensions and function rapidly. The autograft valve shows evidence of growth at greater than 1 year postoperative. The pulmonary autograft may be the ideal valve replacement in children.

The simultaneous occurrence of renal artery stenosis and an aldosteronoma in a patient with hypertension.

The simultaneous occurrence of renovascular hypertension and an adrenocortical adenoma is a rare entity. The case of a 64-year-old woman who underwent an aortorenal bypass graft for renovascular hypertension requiring a multidrug antihypertensive regimen is presented. Persistently elevated blood pressures in the postoperative period prompted further workup for other causes of hypertension. Laboratory evaluation showed hyperaldosteronism and hyporeninemia despite enalapril administration. Abdominal computerized tomography (CT) revealed a left adrenal mass which, on surgical removal, was found to be a cortical adenoma. Subsequently, her antihypertensive therapy has been reduced to a single agent. Previous authors have described only four patients with malignant hypertension who had the rare clinical combination of renal artery stenosis and an aldosteronoma. This case reemphasizes the critical need for a thorough search for other surgically correctable lesions in those patients who remain severely hypertensive after the "definitive" operation.

Forearm hemodynamics at rest and stress in borderline hypertensive adolescents.

Cardiovascular adaptations to mild elevations of blood pressure (BP) may occur early in the development of essential hypertension (EH). We used strain gauge plethysmography to study forearm hemodynamics in adolescents. Ten normotensive males (N) were compared to ten males with borderline hypertension (H). Measurements of forearm blood flow (FBF) were obtained after supine rest, during ten minutes of mental stress (mental arithmetic) and five minutes post stress. Mean arterial pressure (MAP) and forearm vascular resistance (FVR) were calculated. The H maintained higher MAP and FVR (P less than or equal to .05) throughout the study. The vascular response patterns were assessed by comparing the slopes of the linear regression equation FBF = a ln FVR + b (where a = slope and b = intercept). From baseline to stress, the N exhibit a significant change in slope (P less than .05), shifting to a decrease in FVR per unit FBF change. However, the H maintain a constant slope and FVR per unit FBF change remains constant. The study suggests that a primary peripheral vascular abnormality may be present even in the young with marginally elevated BP.