Rationale A growing number of patients with coronary disease have refractory angina. Preclinical and early-phase clinical data suggest that intramyocardial injection of autologous CD34+ cells can improve myocardial perfusion and function. Objective Evaluate the safety and bioactivity of intramyocardial injections of autologous CD34+ cells in patients with refractory angina who have exhausted all other treatment options. Methods and Results In this prospective, double-blind, randomized, phase II study (ClinicalTrials.gov identifier: NCT00300053), 167 patients with refractory angina received 1 of 2 doses (1×105 or 5×105 cells/kg) of mobilized autologous CD34+ cells or an equal volume of diluent (placebo). Treatment was distributed into 10 sites of ischemic, viable myocardium with a NOGA mapping injection catheter. The primary outcome measure was weekly angina frequency 6 months after treatment. Weekly angina frequency was significantly lower in the low-dose group than in placebo-treated patients at both 6 months (6.8±1.1 versus 10.9±1.2, P=0.020) and 12 months (6.3±1.2 versus 11.0±1.2, P=0.035); measurements in the high-dose group were also lower, but not significantly. Similarly, improvement in exercise tolerance was significantly greater in low-dose patients than in placebo-treated patients (6 months: 139±151 versus 69±122 seconds, P=0.014; 12 months: 140±171 versus 58±146 seconds, P=0.017) and greater, but not significantly, in the high-dose group. During cell mobilization and collection, 4.6% of patients had cardiac enzyme elevations consistent with non-ST segment elevation myocardial infarction. Mortality at 12 months was 5.4% in the placebo-treatment group with no deaths among cell-treated patients. Conclusions Patients with refractory angina who received intramyocardial injections of autologous CD34+ cells (105 cells/kg) experienced significant improvements in angina frequency and exercise tolerance. The cell-mobilization and -collection procedures were associated with cardiac enzyme elevations, which will be addressed in future studies.
Background-Understanding the mechanisms of repair and regeneration of the kidney after injury is of great interest because there are currently no therapies that promote repair, and kidneys frequently do not repair adequately. We studied the capacity of human CD34 ϩ hematopoietic stem/progenitor cells (HSPCs) to promote kidney repair and regeneration using an established ischemia/reperfusion injury model in mice, with particular focus on the microvasculature. Methods and Results-Human HSPCs administered systemically 24 hours after kidney injury were selectively recruited to injured kidneys of immunodeficient mice (Jackson Labs, Bar Harbor, Me) and localized prominently in and around vasculature. This recruitment was associated with enhanced repair of the kidney microvasculature, tubule epithelial cells, enhanced functional recovery, and increased survival. HSPCs recruited to kidney expressed markers consistent with circulating endothelial progenitors and synthesized high levels of proangiogenic cytokines, which promoted proliferation of both endothelial and epithelial cells. Although purified HSPCs acquired endothelial progenitor markers once recruited to the kidney, engraftment of human endothelial cells in the mouse capillary walls was an extremely rare event, indicating that human stem cell mediated renal repair is by paracrine mechanisms rather than replacement of vasculature. Conclusions-These studies advance human HSPCs as a promising therapeutic strategy for promoting renal repair after injury. (Circulation. 2010;121:2211-2220.)
This investigation focused on the role played by cold-insoluble globulin (CIg, plasma fibronectin) in monocyte function. Surface-bound CIg mediated a concentration-dependent of human blood monocytes to gelatin-coated surfaces. CIg also mediated the binding of gelatin-coated particles such as latex beads or tanned erythrocytes to surface-bound human monocytes. However, CIg did not mediate particle ingestion. Subfractionated CIg that was highly enriched in monomeric forms (zone II CIg, mol wt 190,000-235,000) was less effective than were fractions enriched in dimeric forms (zone I CIg, mol wt 450,000) in promoting monocyte attachment. Binding of CIg to a gelatin or plastic surface occurred in the absence of divalent cations, but monocyte attachment to CIg-coated surfaces required divalent cations, Mg++ being much more effective than Ca++. Cation-dependent cell attachment was reversible in that bound cells could be released by treatment with EDTA. Serum-mediated binding of monocytes to gelatin-coated plastic dishes was a result of its content of CIg because the binding activity was abolished by removal of CIg from serum, and could be restored by readdition of purified CIg. Treatment of monocytes with trypsin abolished subsequent cell attachment to CIg-gelatin surfaces or particles. Expression of certain other known monocyte membrane receptors (Fc and C3b) was markedly enhanced as a result of CIg-monocyte interaction. These several observations indicate that monocytes bear membrane receptors (termed receptor cold-insoluble globulin) for surface-bound CIg.
Following proteolytic conversion of fibrinogen to fibrin, clot assembly commences with formation of double-stranded fibrils that subsequently branch extensively in forming a three-dimensional network. Plasmin digests of fibrin clots that had first been covalently crosslinked by plasma transglutaminase (factor XIIIa) contained multimeric proteolytic fragments composed of crosslinked outer (D) domains of neighboring fibrin molecules. Two of these were larger than the well-known "D dimer" fragment and corresponded to D trimers and D tetramers, respectively. Whereas D dimers originate from crosslinked D domains at bimolecular junctions within two-stranded fibrils, D trimers and D tetramers evidently arise through crosslinking of contiguous D domains at trimolecular and tetramolecular junctions or at fibril branch points, respectively. Measurement of the widths of fibrils comprising trifunctional branches in thin fiber networks revealed tetramolecular branch points, which are formed by bifurcation of two double-stranded fibrils. In addition, another type of trifunctional structure, which we term the trimolecular branch point, was composed of three double-stranded fibrils. Crosslinking of D domains to form trimers may occur at this type ofjunction. These findings add to our understanding of the crosslinking arrangements that stabilize fibrin clot structure and the ways that fibrin molecules polymerize to form branches in the clot matrix.Following proteolytic conversion of fibrinogen to fibrin, polymer assembly commences with formation of doublestranded fibrils in which fibrin molecules, by virtue of noncovalent intermolecular interactions between outer (D) and central (E) domains (1-6), are arranged in a staggered overlapping manner (7-13) (Fig. 1). Subsequently, lateral fibril associations result in increased fiber thickness (11,(16)(17)(18), which is believed to account for interfibril connections and the trifunctional branching structures that comprise the three-dimensional matrix (11,(18)(19)(20)(21). In the presence of plasma transglutaminase (factor XIIIa) and Ca2', fibrin molecules undergo covalent crosslinking by formation of E-(y-glutamyl)lysine [E-('y-Glu)Lys] isopeptide bonds (22,23). Intermolecular crosslinking between D domains forms dimers (24), which occur as reciprocal bridges between a lysine at position 406 of one 'y chain and a glutamine at position 398 or 399 of another (25)(26)(27)(28). In addition, slower intermolecular crosslinking among a chains creates oligomers and larger a-chain polymers (29-31).Plasmin digestion of crosslinked fibrin results in early release of crosslink-containing a-chain segments from core structures (32-36). Thus, their existence in fibrin does not contribute significantly to the structure of major intermediate or terminal plasmin core fragments. In contrast, the intermolecular e-(y-Glu)Lys y-chain bonds result in degradation products unique to crosslinked fibrin, of which the bimolecular fragment, "D dimer," is the most abundant and best characterized (32, 37-39...
As assessed by electron microscopy, the reported shape of the plasma fibronectin molecule ranges from that of a compact particle to an elongated, rod-like structure . In this study, we evaluated the effects of solution and surface conditions on fibronectin shape . Freeze-dried, unstained human plasma fibronectin molecules deposited at pH 7 .0-7 .4 onto carbon films and examined by scanning transmission electron microscopy appeared relatively compact and pleiomorphic, with approximate average dimensions of 24 nm x 16 nm . Negatively stained molecules also had a similar shape but revealed greater detail in that we observed irregular, yarn-like structures . Glutaraldehyde-induced intramolecular cross-linking did not alter the appearance of plasma fibronectin . Molecules deposited at pH 2 .8, pH 9 .3, or after succinylation were less compact than those deposited at neutral pH . In contrast, fibronectin molecules sprayed onto mica surfaces at pH 7, rotary shadowed, and examined by transmission electron microscopy were elongated and nodular with a contour length of 120-130 nm .Sedimentation velocity experiments and electron microscopic observations indicate that fibronectin unfolds when it is succinylated, when the ionic strength is raised at pH 7, or when the pH is adjusted to 9 .3 or 2 .8 . Greater unfolding is observed at pH 2 .8 at low ionic strength (<0.01) compared with material at that pH in 0.15 M NaCl solution . We conclude that (a) the shape assumed by the fibronectin molecule can be strongly affected by solution conditions and by deposition onto certain surfaces ; and that (b) the images of fibronectin seen by scanning transmission electron microscopy at neutral pH on carbon film are representative of molecules in physiologic solution .Fibronectin' is the term generally used to describe a family of structurally and immunologically related high molecular weight glycoproteins that are found on many cell surfaces, in extracellular fluids, in connective tissues, and in most basement membranes. One of the universal features of all fibronectin molecules is the presence of distinct binding regions, each typically having a selective affinity for one or another of several different macromolecules such as collagen, fibrin(ogen), other fibronectin molecules, heparin, actin, DNA, and others (1-4). Binding regions also exist for certain mammalian cells (e.g ., fibroblasts, monocytes) and for certain bacteria (e.g ., Staphylococcus aureus) (1-4). These multiple binding sites on fibronectin evidently mediate several biologic "adhesive" activities such as cell to cell adhesion, cell to substrate attachment and spreading, collagen attachment to mononuclear phagocytes and/or to filamentous matrices during formation, and remodeling of connective tissue.The major circulating form of human plasma fibronectin is a dimer of molecular weight 450,000 (5) . The two chains are linked together by disulfide bridges near each carboxy terminus (1-4, 6) . The far ultraviolet circular dichroism spectrum gives little evidence of hi...
Release of fibrinopeptide B from fibrinogen by copperhead venom procoagulant enzyme results in a form of fibrin (beta-fibrin) with weaker self-aggregation characteristics than the normal product (alpha beta-fibrin) produced by release of fibrinopeptides A (FPA) and B (FPB) by thrombin. We investigated the ultrastructure of these two types of fibrin as well as that of beta-fibrin prepared from fibrinogen Metz (A alpha 16 Arg----Cys), a homozygous dysfibrinogenemic mutant that does not release FPA. At 14 degrees C and physiologic solvent conditions (0.15 mol/L of NaCl, 0.015 mol/L of Tris buffer pH 7.4), the turbidity (350 nm) of rapidly polymerizing alpha beta-fibrin (thrombin 1 to 2 U/mL) plateaued in less than 6 min and formed a “coarse” matrix consisting of anastomosing fiber bundles (mean diameter 92 nm). More slowly polymerizing alpha beta-fibrin (thrombin 0.01 and 0.001 U/mL) surpassed this turbidity after greater than or equal to 60 minutes and concomitantly developed a network of thicker fiber bundles (mean diameters 118 and 186 nm, respectively). Such matrices also contained networks of highly branched, twisting, “fine” fibrils (fiber diameters 7 to 30 nm) that are usually characteristic of matrices formed at high ionic strength and pH. Slowly polymerizing beta-fibrin, like slowly polymerizing alpha beta-fibrin, displayed considerable quantities of fine matrix in addition to an underlying thick cable network (mean fiber diameter 135 nm), whereas rapidly polymerizing beta-fibrin monomer was comprised almost exclusively of wide, poorly anastomosed, striated cables (mean diameter 212 nm). Metz beta-fibrin clots were more fragile than those of normal beta-fibrin and were comprised almost entirely of a fine network. Metz fibrin could be induced, however, to form thick fiber bundles (mean diameter 76 nm) in the presence of albumin at a concentration (500 mumol/L) in the physiologic range and resembled a Metz plasma fibrin clot in that regard. The diminished capacity of Metz beta-fibrin to form thick fiber bundles may be due to impaired use or occupancy of a polymerization site exposed by FPB release. Our results indicate that twisting fibrils are an inherent structural feature of all forms of assembling fibrin, and suggest that mature beta-fibrin or alpha beta-fibrin clots develop from networks of thin fibrils that have the ability to coalesce to form thicker fiber bundles.
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