Expression levels of insulin receptor substrate-1 modulate the osteoblastic differentiation of mesenchymal stem cells and osteosarcoma cells.

The insulin-like growth factor-1 system, including its critical mediator insulin receptor substrate-1 (IRS-1), is involved in regulating osteosarcoma (OS) cell proliferation or differentiation. The aim of this study is to define the role of IRS-1 in OS cells by assessing the contribution of IRS-1 in the differentiation of human and murine OS cell lines and mouse mesenchymal stem cells (MSCs) and found that the basal level of IRS-1 is important for the initiation of differentiation. Both down-regulation and over-expression of IRS-1 inhibited osteoblastic differentiation. In vivo studies showed that OS cells over-expressing IRS-1 have increased metastatic potential and tumor growth. The proteasome inhibitor MG-132 led to an increase in IRS-1 protein level that inhibited osteoblastic differentiation, suggesting a role for proteasomal regulation in maintaining the appropriate expression level of IRS-1. Thus, precise regulation of IRS-1 expression level is critical for determining the differentiating capacity of MSCs and OS cells, and that derangement of IRS-1 levels can be a critical step in OS transformation.

Systemically delivered insulin-like growth factor-I enhances mesenchymal stem cell-dependent fracture healing.

In this study, we examined the effectiveness of systemic subcutaneous delivery of recombinant Insulin-like growth factor (IGF)-I concurrently with primary cultured bone marrow-derived mesenchymal stem cell (MSC) transplant on fracture repair. We found that the fracture callus volume increased in mice with a stabilized tibia fracture that received IGF-I+MSC when compared with that in either untreated or MSC alone treated mice. In evaluating the callus tissue components, we found that the soft and new bone tissue volumes were significantly increased in IGF-I+MSC recipients. Histological and in-situ hybridization analyses confirmed a characteristic increase of newly forming bone in IGF-I+MSC recipients and that healing progressed mostly through endochondral ossification. The increase in soft and new bone tissue volumes correlated with increased force and toughness as determined by biomechanical testing. In conclusion, MSC transplant concurrent with systemic delivery of IGF-I improves fracture repair suggesting that IGF-I+MSC could be a novel therapeutic approach in patients who have inadequate fracture repair.

Mesenchymal stem cells expressing insulin-like growth factor-I (MSCIGF) promote fracture healing and restore new bone formation in Irs1 knockout mice: analyses of MSCIGF autocrine and paracrine regenerative effects.

Failures of fracture repair (nonunions) occur in 10% of all fractures. The use of mesenchymal stem cells (MSC) in tissue regeneration appears to be rationale, safe, and feasible. The contributions of MSC to the reparative process can occur through autocrine and paracrine effects. The primary objective of this study is to find a novel mean, by transplanting primary cultures of bone marrow-derived MSCs expressing insulin-like growth factor-I (MSC(IGF)), to promote these seed-and-soil actions of MSC to fully implement their regenerative abilities in fracture repair and nonunions. MSC(IGF) or traceable MSC(IGF)-Lac-Z were transplanted into wild-type or insulin-receptor-substrate knockout (Irs1(-/-)) mice with a stabilized tibia fracture. Healing was assessed using biomechanical testing, microcomputed tomography (µCT), and histological analyses. We found that systemically transplanted MSC(IGF) through autocrine and paracrine actions improved the fracture mechanical strength and increased new bone content while accelerating mineralization. We determined that IGF-I adapted the response of transplanted MSC(IGF) to promote their differentiation into osteoblasts. In vitro and in vivo studies showed that IGF-I-induced osteoglastogenesis in MSCs was dependent of an intact IRS1-PI3K signaling. Furthermore, using Irs1(-/-) mice as a nonunion fracture model through altered IGF signaling, we demonstrated that the autocrine effect of IGF-I on MSC restored the fracture new bone formation and promoted the occurrence of a well-organized callus that bridged the gap. A callus that was basically absent in Irs1(-/-) left untransplanted or transplanted with MSCs. We provided evidence of effects and mechanisms for transplanted MSC(IGF) in fracture repair and potentially to treat nonunions.

Regenerative effects of transplanted mesenchymal stem cells in fracture healing.

Mesenchymal stem cells (MSC) have a therapeutic potential in patients with fractures to reduce the time of healing and treat nonunions. The use of MSC to treat fractures is attractive for several reasons. First, MSCs would be implementing conventional reparative process that seems to be defective or protracted. Secondly, the effects of MSCs treatment would be needed only for relatively brief duration of reparation. However, an integrated approach to define the multiple regenerative contributions of MSC to the fracture repair process is necessary before clinical trials are initiated. In this study, using a stabilized tibia fracture mouse model, we determined the dynamic migration of transplanted MSC to the fracture site, their contributions to the repair process initiation, and their role in modulating the injury-related inflammatory responses. Using MSC expressing luciferase, we determined by bioluminescence imaging that the MSC migration at the fracture site is time- and dose-dependent and, it is exclusively CXCR4-dependent. MSC improved the fracture healing affecting the callus biomechanical properties and such improvement correlated with an increase in cartilage and bone content, and changes in callus morphology as determined by micro-computed tomography and histological studies. Transplanting CMV-Cre-R26R-Lac Z-MSC, we found that MSCs engrafted within the callus endosteal niche. Using MSCs from BMP-2-Lac Z mice genetically modified using a bacterial artificial chromosome system to be beta-gal reporters for bone morphogenic protein 2 (BMP-2) expression, we found that MSCs contributed to the callus initiation by expressing BMP-2. The knowledge of the multiple MSC regenerative abilities in fracture healing will allow design of novel MSC-based therapies to treat fractures.

Transcutaneous energy transmission for mechanical circulatory support systems: history, current status, and future prospects.

A totally implantable mechanical circulatory support system would be very desirable for destination therapy. However, implanting all components of a pulsatile total artificial heart (TAH) or left ventricular assist device (LVAD) is complex because of the requirement for a continuous electrical power supply and the need for volume compensation. Implantable compliance chambers were developed for early LVAD designs, and although they functioned properly during initial laboratory tests, air loss by diffusion and the development of fibrous tissue around the sac eventually rendered them ineffective. Because these problems have not yet been overcome, volume displacement LVADs are currently designed with either a direct communication to an external drive console or an atmospheric vent. Transcutaneous energy transmission systems (TETSs) were also developed, but because the skin was being penetrated for volume compensation, it seemed more efficient to transmit electrical power through wires incorporated into the venting apparatus. More recently, TETSs were used clinically for both a pulsatile TAH and LVAD in a small number of patients, but for reasons unrelated to the TETS, neither of these devices is presently in use. Because the newer continuous-flow LVADs do not require a compliance chamber, they present a potential future application for TETS technology, because infections of the percutaneous tube continue to be one of the most important limitations of long-term circulatory support. A totally implantable LVAD with an incorporated TETS for destination therapy could become an important advance in the treatment of end-stage heart failure.

Subcellular localization of IRS-1 in IGF-I-mediated chondrogenic proliferation, differentiation and hypertrophy of bone marrow mesenchymal stem cells.

Bone marrow derived mesenchymal stem cells (BM-MSC) can differentiate into chondrocytes. Understanding the mechanisms and growth factors that control the MSC stemness is critical to fully implement their therapeutic use in cartilage diseases. The activated type 1 insulin-like growth factor receptor (IGF-IR), interacting with the insulin receptor substrate-1 (IRS-1), can induce cancer cell proliferation and transformation. In cancer or transformed cells, IRS-1 has been shown to localize in the cytoplasm where it activates the canonical Akt pathway, as well as in the nucleus where it binds to nuclear proteins. We have previously demonstrated that IGF-I has distinct time-dependent effect on primary BM-MSC chondrogenic pellets: initially (2-day culture), IGF-I induces proliferation; subsequently, IGF-I promotes chondrocytic differentiation (7-day culture). In the present study, by using MSC from the BM of IRS-1(- / - ) mice we show that IRS-1 mediates almost 50% of the IGF-I mitogenic response and the MAPK-MEK/ERK signalling accounts for the other 50%. After stimulation with IGF-I, we found that in 2-day old human and mouse derived BM-MSC pellets, IRS-1 (total and phosphorylated) is nuclearly localized and that proliferation prevails over differentiation. The IGF-I mitogenic effect is Akt-independent. In 7-day MSC pellets, IGF-I stimulates the chondrogenic differentiation of MSC into chondrocytes, pre-hypertrophic and hypertrophic chondrocytes and IRS-1 accumulates in the cytoplasm. IGF-I-dependent differentiation is exclusively Akt-dependent. Our data indicate that in the physiologically relevant model of primary cultured MSC, IGF-I induces a temporally regulated nuclear or cytoplasmic localization of IRS-1 that correlate with the transition from proliferation to chondrogenic differentiation.

Reducing aluminum dust explosion hazards: case study of dust inerting in an aluminum buffing operation.

Metal powders or dusts can represent significant dust explosion hazards in industry, due to their relatively low ignition energy and high explosivity. The hazard is well known in industries that produce or use aluminum powders, but is sometimes not recognized by facilities that produce aluminum dust as a byproduct of bulk aluminum processing. As demonstrated by the 2003 dust explosion at aluminum wheel manufacturer Hayes Lemmerz, facilities that process bulk metals are at risk due to dust generated during machining and finishing operations [U.S. Chemical Safety and Hazard Investigation Board, Investigation Report, Aluminum Dust Explosion Hayes Lemmerz International, Inc., Huntington, Indiana, Report No. 2004-01-I-IN, September 2005]. Previous studies have shown that aluminum dust explosions are more difficult to suppress with flame retardants or inerting agents than dust explosions fueled by other materials such as coal [A.G. Dastidar, P.R. Amyotte, J. Going, K. Chatrathi, Flammability limits of dust-minimum inerting concentrations, Proc. Saf. Progr., 18-1 (1999) 56-63]. In this paper, an inerting method is discussed to reduce the dust explosion hazard of residue created in an aluminum buffing operation as the residue is generated. This technique reduces the dust explosion hazard throughout the buffing process and within the dust collector systems making the process inherently safer. Dust explosion testing results are presented for process dusts produced during trials with varying amounts of flame retardant additives.

Simultaneous insertion of a left ventricular assist system and repair of an ascending aortic dissection.

Operative methods for repairing ascending aortic dissections and for implanting left ventricular assist systems have been thoroughly presented in the medical literature. Only a few reports, however, describe the concomitant performance of these procedures in 1 patient. We report the repair of an acute ascending aortic dissection with simultaneous placement of a long-term left ventricular assist system. One week earlier, the patient had undergone emergent coronary artery bypass grafting and short-term postcardiotomy ventricular assistance when he could not be weaned from cardiopulmonary bypass. By creating a graft-to-graft anastomosis on the bench during cooling of the patient on cardiopulmonary bypass, we were able to shorten to 21 minutes the period of hypothermic circulatory arrest required during ascending aortic dissection repair. The procedures were completed successfully. However, the patient developed pneumonia and sepsis during his extended hospital stay and died of multiorgan failure 5 weeks postoperatively.

Initial clinical experience with the HeartMate II axial-flow left ventricular assist device.

The redesigned HeartMate II, an axial-flow left ventricular assist device, is simpler, smaller, and easier to operate than are pulsatile pumps. These design characteristics should make the HeartMate II more reliable and durable and broaden the eligible population base. We implanted the HeartMate II in 43 patients (average age, 42 yr). The indication for use was bridge-to-heart transplantation in 26 patients and destination therapy in 17. The average duration of device support was 258 days (range, 1-761 days), and cumulative duration, more than 31 patient-years. Hemodynamic function improved in all patients during support. By 48 hours after implantation, the mean cardiac index had increased from 1.9+/-0.27 L/(min.m(2)) (baseline) to 3.5+/-0.8 L/(min.m(2)), and the pulmonary capillary wedge pressure had decreased from 24.8+/-11 mmHg to 18.5+/-5.3 mmHg. Of the 43 patients, 35 were discharged from the hospital. Support is ongoing in 27 patients (longest duration, >700 days). Nine patients died during support. Four patients had sufficient heart recovery to undergo pump explantation. Three patients underwent transplantation. One patient underwent device replacement after the pump driveline was fractured in a skateboarding accident; the device was removed in another patient because of a pump-pocket infection after 749 days of support. Of the 10 patients in whom the HeartMate II replaced a failed HeartMate I, 8 were discharged from the hospital. We have seen excellent results with use of the HeartMate II. Functional status and quality of life have greatly improved in patients who survived the perioperative period.

Management of air embolism during HeartMate XVE exchange.

Air embolism is a rare and usually fatal complication of major cardiac surgery. We present a case in which a 45-year-old man supported by a HeartMate(R) XVE left ventricular assist device required a pump exchange due to failure of the device motor. During pump dissection, a massive amount of air entered the systemic circulation. Urgent cannulation for cardiopulmonary bypass was performed, and cardiopulmonary bypass was initiated, followed by profound hypothermia, circulatory arrest, retrograde cerebral perfusion, retrograde coronary sinus perfusion, and then barbiturate coma and steroid therapy. The HeartMate XVE left ventricular assist device was removed, and a HeartMate II was implanted. After 5 days, the patient awoke with left hemiparesis, which nearly resolved with aggressive physical therapy. Forty-four days after the pump exchange operation, the patient was discharged from the hospital with only mild left hemiparesis. Exposure of the left ventricular assist device or its external components requires careful monitoring, because air can enter the pump-particularly in a hypovolemic patient. Rapid response after massive air entry into the left ventricular assist device system, as in our patient, can result in a successful outcome.

Myocardial perfusion as assessed by positron emission tomography during long-term mechanical circulatory support.

Although mechanical circulatory support (MCS) can improve myocardial function in patients with advanced heart failure, its effects on relative myocardial perfusion are unclear. Using positron emission tomographic imaging techniques, the authors assessed relative myocardial perfusion in patients with ischemic or idiopathic cardiomyopathy who were receiving chronic MCS with a left ventricular assist device (pulsatile HeartMate [n = 2] [Thoratec Corporation, Pleasanton, CA] or nonpulsatile Jarvik 2000 [n = 4] [Jarvik Heart, Inc., New York, NY]). Relative myocardial perfusion was compared at lower and higher levels of MCS (50 vs. 100 - 110 ejections/min for the HeartMate and 8000 vs. 12,000 rpm for the Jarvik 2000). The size and severity of perfusion defects at rest and after dipyridamole stress were measured objectively and subjectively by computer algorithms and visual inspection, respectively. Relative myocardial perfusion increased > 5% from baseline in only one of six patients when MCS was increased. No change in relative myocardial perfusion of > 5% was seen in any of the other five patients, even after subsequent dipyridamole stress positron emission tomographic imaging. These pilot study findings suggest that the decreased metabolic requirements induced by ventricular unloading correspondingly decreased blood flow requirements to physiologically inactive myocardium.

Saphenous vein graft flow during left ventricular assistance with an axial-flow pump.

The effects of continuous-flow support on bypass graft flow have not been quantified clinically. Continuous-flow left ventricular assist devices unload the left ventricle throughout the cardiac cycle, which narrows pulse pressure and converts passive left ventricular filling during diastole to active flow throughout the cardiac cycle. We report the case of a 63-year-old man with severe congestive heart failure who underwent coronary artery bypass grafting and was supported with an axial-flow pump. In this patient, saphenous vein graft flow during left ventricular assistance provided adequate coronary perfusion.

Clinical experience with the TandemHeart percutaneous ventricular assist device.

The TandemHeart percutaneous ventricular assist device can be used to support patients in cardiogenic shock (until cardiac recovery occurs or as a bridge to definitive therapy) or as a temporary application during high-risk coronary interventions. The TandemHeart is a left atrial-to-femoral artery bypass system comprising a transseptal cannula, arterial cannulae, and a centrifugal blood pump. The pump can deliver flow rates up to 4.0 L/min at a maximum speed of 7500 rpm. From May 2003 through May 2005, the TandemHeart was used to support 18 patients (11 in cardiogenic shock and 7 undergoing high-risk percutaneous transluminal coronary angioplasty). The patients in cardiogenic shock were supported for a mean of 88.8 +/- 74.3 hours (range, 4-264 hr) at a mean pump flow rate of 2.87 +/- 0.56 L/min (range, 1.8-3.5 L/min). The mean cardiac index improved from 1.57 +/- 0.31 L/min/m2 before support to 2.60 +/- 0.34 L/min/m2 during support. The mean duration of support for the high-risk percutaneous transluminal coronary angioplasty patients was 5.5 +/- 8.3 hours (range, 1-24 hr). The mean flow rate was 2.42 +/- 0.55 L/min (range, 1.5-3.0 L/ min). The overall 30-day survival rate was 61%. In our experience, the TandemHeart device was easy to insert and provided a means either to cardiac recovery or to continued support with an implantable left ventricular assist device.

Catheterization of the AbioCor implantable replacement heart: evaluation of the unique physiology created by the device.

We performed the 1st catheterization of an AbioCor implantable replacement heart, in a patient who had developed high right-sided pump pressures, to determine whether the high pressures were caused by graft kinking or obstruction.

Reversal of myocyte hypertrophy by ventricular unloading: cardiac improvement without adrenergic receptor up-regulation and relocalization.

In previous studies, we found that the improved contractile ability of cardiac myocytes from patients who have had left ventricular assist device (LVAD) support was due to a number of beneficial changes, most notably in calcium handling (increased sarcoplasmic reticulum calcium binding and uptake), improved integrity of cell membranes due to phospholipid reconstruction (reduced lysophospholipid content), and an upregulation of adrenoreceptors (increased adrenoreceptor numbers). However, in the case presented here, there was no increase in adrenoreceptor number, which is something that we usually find in core tissue at the time of LVAD removal or organ transplantation; also, there was no homogeneous postassist device receptor distribution. However, the patient was well maintained for 10 months following LVAD implantation, until a donor organ was available, regardless of the lack of adrenoreceptor improvement. We conclude from these studies that cardiac recovery is the result of the initiation of multiple repair mechanisms, and that the lack of expected changes, in this case increased adrenoreceptors, is not always an accurate indicator of anticipated outcome. We suggest that interventions and strategies have to consider multiple, beneficial changes due to unloading and target a number of biochemical and structural areas to produce improvement, even if not all of these improvements occur.

Initial clinical experience with the Jarvik 2000 implantable axial-flow left ventricular assist system.

BACKGROUND: Implantable left ventricular assist systems (LVASs) are used for bridging to transplantation, bridging to myocardial improvement, and for permanent circulatory support. Conventional implantable systems have inherent limitations that increase morbidity during support. In contrast, small, efficient, axial-flow pumps, which have been under development for the past decade, have the potential to improve the length and quality of life in patients with severe heart failure. Methods and Results- To assess the safety and clinical utility of the Jarvik 2000, we implanted this device in 10 transplant candidates (mean age 51.3 years) in New York Heart Association (NYHA) class IV. Implantation was achieved through a left thoracotomy during partial cardiopulmonary bypass. The mean support period was 84 days. Within 48 hours postoperatively, the cardiac index increased 43%, pulmonary capillary wedge pressure decreased 52%, systemic vascular resistance decreased significantly, and inotropic support became unnecessary. Eight patients underwent physical rehabilitation and returned to NYHA class I. Their left ventricular dimensions, cardiothoracic ratios, and pressure-volume loop analyses showed good left ventricular unloading. Seven patients underwent transplantation and 3 died during support. No device thrombosis was observed at explantation. CONCLUSIONS: The Jarvik 2000 functions as a true assist device by partially unloading the left ventricle, thereby optimizing the patient's hemodynamics. Our preliminary results indicate that this LVAS may safely provide circulatory assistance for heart transplant candidates.

Gastrointestinal bleeding from arteriovenous malformations in patients supported by the Jarvik 2000 axial-flow left ventricular assist device.

The long-term effects of axial-flow mechanical circulatory support in humans are unclear. We report 3 cases of chronic gastrointestinal bleeding after implantation of a Jarvik 2000 axial-flow left ventricular assist device. The bleeding was refractory to aggressive management and in 2 cases resolved only after orthotopic cardiac transplantation.

Direct thrombolytic therapy for intraventricular thrombosis in patients with the Jarvik 2000 left ventricular assist device.

One of the complications that can occur with continuous, axial-flow left ventricular assist devices (LVADs) is thrombosis within the left ventricle, adjacent to the device's inflow conduit, which may cause inflow obstruction and recurrent heart failure. We describe 2 cases in which we used a catheter to continuously infuse recombinant tissue plasminogen activator (tPA) into the left ventricle until signs of successful thrombolysis was achieved. By monitoring the result and administering only as much tPA as necessary to achieve thrombolysis, we were able to successfully lyse the obstructing thrombus with a minimal dose of tPA without causing any significant bleeding problems. This technique may be useful for managing this potentially serious complication while minimizing the risk of treatment.

Role of B-type natriuretic peptide and effect of nesiritide after total cardiac replacement with the AbioCor total artificial heart.

Endogenous B-type natriuretic peptide (BNP) is thought to be produced in the cardiac ventricles. After sub-total cardiectomy and implantation of a total artificial heart (TAH), the abrupt withdrawal of BNP impairs renal function despite normal hemodynamic variables. We hypothesized that abrupt withdrawal of endogenous BNP may impair renal function and volume homeostasis and BNP may have a direct renal influence unrelated to its cardiovascular effect. Nesiritide infusion should be supplemented in the interim and weaned slowly until BNP levels normalize, which suggests that BNP is produced in tissues other than the cardiac ventricles.

Nitric oxide versus prostaglandin E1 for reduction of pulmonary hypertension in heart transplant candidates.

BACKGROUND: We sought to directly compare the effects of prostaglandin E1 (PGE1) and nitric oxide (NO) in testing for pulmonary hypertension reversibility in heart transplant candidates. METHODS: We included 19 heart transplant candidates who fulfilled at least 1 of 3 criteria: pulmonary vascular resistance (PVR) of >4 Wood units; transpulmonary gradient (TPG) of >12 mmHg; or systolic pulmonary artery pressure (PAP) of >60 mmHg. Patients randomly received either PGE1 (0.05, 0.2 and 0.5 microg/kg/min) or NO (40, 60 and 80 ppm) and were crossed-over to the second medication after receiving the maximal dose of the first. RESULTS: With PGE1, TPG decreased by 21% (baseline 20.3 +/- 6.8 mmHg; final 16.0 +/- 7.0 mmHg) compared to a 34% decrease with NO (baseline 20.8 +/- 6.2 mmHg; final 13.8 +/- 5.4 mmHg) (p = 0.13). PVR decreased by 42% with PGE1 (baseline 6.2 +/- 4.0 Wood units; final 3.6 +/- 1.8 Wood units) and by 47% with NO (baseline 6.0 +/- 3.9 Wood units; final 3.2 +/- 1.6 Wood units) (p = 0.87). Mean systemic pressure decreased with PGE1 (baseline 76.1 +/- 10.5 mmHg; final 69.4 +/- 12.2 mmHg; -9%) but not with NO administration (baseline 70.2 +/- 14.7 mmHg; final 71.6 +/- 10.9 mmHg; +2%) (p = 0.01). TPG was lowered to <12 mmHg in 14 patients. Of these, 6 (46%) responded to both PGE1 and NO, 4 (27%) responded only to PGE1, and 4 (27%) responded only to NO. CONCLUSIONS: The effects of PGE1 and NO on pulmonary hypertension are comparable, with PGE1 having more systemic hypotensive effects. Due to variability of patient responses, we recommend multiple rather than single-agent pharmacologic testing for the reversibility of pulmonary hypertension.