A Biomarker for Predicting Responsiveness to Stem Cell Therapy Based on Mechanism-of-Action: Evidence from Cerebral Injury.

To date, no stem cell therapy has been directed to specific recipients-and, conversely, withheld from others-based on a clinical or molecular profile congruent with that cell's therapeutic mechanism-of-action (MOA) for that condition. We address this challenge preclinically with a prototypical scenario: human neural stem cells (hNSCs) against perinatal/neonatal cerebral hypoxic-ischemic injury (HII). We demonstrate that a clinically translatable magnetic resonance imaging (MRI) algorithm, hierarchical region splitting, provides a rigorous, expeditious, prospective, noninvasive "biomarker" for identifying subjects with lesions bearing a molecular profile indicative of responsiveness to hNSCs' neuroprotective MOA. Implanted hNSCs improve lesional, motor, and/or cognitive outcomes only when there is an MRI-measurable penumbra that can be forestalled from evolving into necrotic core; the core never improves. Unlike the core, a penumbra is characterized by a molecular profile associated with salvageability. Hence, only lesions characterized by penumbral > core volumes should be treated with cells, making such measurements arguably a regenerative medicine selection biomarker.

Long-term magnetic resonance imaging of stem cells in neonatal ischemic injury.

OBJECTIVE: Quantitative magnetic resonance imaging (MRI) can serially and noninvasively assess the degree of injury in rat pup models of hypoxic ischemic injury (HII). It can also noninvasively monitor stem cell migration following iron oxide prelabeling. Reports have shown that neural stem cells (NSCs) may help mediate neuroprotection or stimulate neuroreparative responses in adult and neonatal models of ischemic injury. We investigated the ability of high-field MRI to monitor and noninvasively quantify the migration, proliferation, and location of iron oxide-labeled NSCs over very long time periods (58 weeks) in real time while contemporaneously correlating this activity with the evolving severity and extent of neural damage. METHODS: Labeled clonal murine NSCs (mNSCs) were implanted 3 days after unilateral HII in 10-day-old rat pups into the contralateral striatum or ventricle. We developed methods for objectively quantifying key aspects of dynamic NSC behavior (eg, viability; extent, and speed of migration; degree of proliferation; extent of integration into host parenchyma). MRI images were validated with histological and immunohistochemical assessments. RESULTS: mNSCs rapidly migrated (100 µm/day) to the lesion site. Chains of migrating NSCs were observed in the corpus callosum. In pups subjected to HII, though not in intact control animals, we observed a 273% increase in the MR-derived volume of mNSCs 4 weeks after implantation (correlating with the known proliferative behavior of endogenous and exogenous NSCs) that slowly declined over the 58-week time course, with no adverse consequences. Large numbers of now quiescent mNSCs remained at the site of injury, many retaining their iron oxide label. INTERPRETATION: Our studies demonstrate that MRI can simultaneously monitor evolving neonatal cerebral injury as well as NSC migration and location. Most importantly, it can noninvasively monitor proliferation dynamically for prolonged time periods. To be able to pursue clinical trials in newborns using stem cell therapies it is axiomatic that safety be insured through the long-term real time monitoring of cell fate and activity, particularly with regard to observing unanticipated risks to the developing brain. This study supports the feasibility of reliably using MRI for this purpose.

Molecular determinants of frequency dependence and Ca2+ potentiation of verapamil block in the pore region of Cav1.2.

Verapamil block of Ca(v)1.2 is frequency-dependent and potentiated by Ca(2+). We examined the molecular determinants of these characteristics using mutations that effect Ca(2+) interactions with Ca(v)1.2. Mutant and wild-type Ca(v)1.2 channels were transiently expressed in tsA 201 cells with beta(1b) and alpha(2)delta subunits. The four conserved glutamates that compose the Ca(2+) selectivity filter in Ca(v)1.2 were mutated to Gln (E363Q, E709Q, E1118Q, E1419Q) and the adjacent conserved threonine in each domain was mutated to Ala (T361A, T707A, T1116A, T1417A). The L-type-specific residues in the domain III pore region (F1117G) and the C-terminal tail (I1627A) were also mutated and assayed for block by verapamil using whole-cell voltage-clamp recordings in 10 mM Ba(2+) or 10 mM Ca(2+). In Ba(2+), none of the pore-region mutations reduced the fraction of current blocked by 30 microM verapamil at 0.05 Hz stimulation. However, all of the pore-region mutations abolished Ca(2+) potentiation of verapamil block at 0.05 Hz. The T1116A, F1117G, E1118Q, and E1419Q mutations all significantly reduced frequency-dependent verapamil block (1-Hz stimulation) in both Ba(2+) and Ca(2+). The I1627A mutation, which disrupts Ca(2+)-dependent inactivation, increased the fraction of closed channels blocked by 30 microM verapamil in Ba(2+) but did not affect frequency-dependent block in Ba(2+) or Ca(2+). Our data suggest that the pore region of domain III may contribute to a high affinity verapamil binding site accessed during 1-Hz stimulation and that Ca(2+) binding to multiple sites may be required for potentiation of verapamil block of closed channels.

Differential antifungal and calcium channel-blocking activity among structurally related plant defensins.

Plant defensins are a family of small Cys-rich antifungal proteins that play important roles in plant defense against invading fungi. Structures of several plant defensins share a Cys-stabilized alpha/beta-motif. Structural determinants in plant defensins that govern their antifungal activity and the mechanisms by which they inhibit fungal growth remain unclear. Alfalfa (Medicago sativa) seed defensin, MsDef1, strongly inhibits the growth of Fusarium graminearum in vitro, and its antifungal activity is markedly reduced in the presence of Ca(2+). By contrast, MtDef2 from Medicago truncatula, which shares 65% amino acid sequence identity with MsDef1, lacks antifungal activity against F. graminearum. Characterization of the in vitro antifungal activity of the chimeras containing portions of the MsDef1 and MtDef2 proteins shows that the major determinants of antifungal activity reside in the carboxy-terminal region (amino acids 31-45) of MsDef1. We further define the active site by demonstrating that the Arg at position 38 of MsDef1 is critical for its antifungal activity. Furthermore, we have found for the first time, to our knowledge, that MsDef1 blocks the mammalian L-type Ca(2+) channel in a manner akin to a virally encoded and structurally unrelated antifungal toxin KP4 from Ustilago maydis, whereas structurally similar MtDef2 and the radish (Raphanus sativus) seed defensin Rs-AFP2 fail to block the L-type Ca(2+) channel. From these results, we speculate that the two unrelated antifungal proteins, KP4 and MsDef1, have evolutionarily converged upon the same molecular target, whereas the two structurally related antifungal plant defensins, MtDef2 and Rs-AFP2, have diverged to attack different targets in fungi.

Molecular determinants of Ca2+ potentiation of diltiazem block and Ca2+-dependent inactivation in the pore region of cav1.2.

Diltiazem block of Cav1.2 is frequency-dependent and potentiated by Ca2+. We examined the molecular determinants of these characteristics using mutations that affect Ca2+ interactions with Cav1.2. Mutant and wild-type (WT) Cav1.2 channels were transiently expressed in tsA 201 cells with beta1b and alpha2delta subunits. The four conserved glutamates that compose the Ca2+ selectivity filter in Cav1.2 were mutated to Gln (E363Q, E709Q, E1118Q, E1419Q), and each single mutant was assayed for block by diltiazem using whole-cell voltage-clamp recordings in either 10 mM Ba2+ or 10 mM Ca2+. In Ba2+, none of the mutations affected the potency of diltiazem block of closed channels (0.05 Hz stimulation). However, frequency-dependent block (1Hz stimulation) was eliminated in the mutant E1419Q (domain IV), which recovered more rapidly than WT channels from inactivated channel block. Potentiation of diltiazem block of closed Cav1.2 channels in Ca2+ was abolished in the E1118Q, F1117G (domain III), and E1419Q mutants. Frequency-dependent block in Ca2+ was reduced compared with WT Cav1.2 in the F1117G, E1118Q, and E1419Q mutants. The C-terminal tail IQ domain mutation I1627A, which disrupts Ca2+ dependent inactivation, enhanced diltiazem block of closed channels in Ba2+. We conclude that, in Ba2+, E1419 slows recovery from diltiazem block of depolarized Cav1.2 channels, but in Ca2+, E1118, E1419, and F1117 form a Ca2+ binding site that mediates the potentiation of diltiazem block of both closed and inactivated Cav1.2 channels. Furthermore, Ca2+-dependent inactivation, which is impaired in E709Q, E1118Q, E1419Q, and I1627A, is not required for Ca2+ potentiation of diltiazem block.

Ca v 1.3 is preferentially coupled to glucose-stimulated insulin secretion in the pancreatic beta-cell line INS-1.

L-Type Ca(2+) channel blockers inhibit glucose and KCl-stimulated insulin secretion by pancreatic beta cells. However, the role of the two distinct L-type channels expressed by beta cells, Ca(v)1.2 and Ca(v)1.3, in this process is not clear. Therefore, we stably transfected INS-1 cells with two mutant channel constructs, Ca(v)1.2DHPi or Ca(v)1.3 DHPi. Whole-cell patch-clamp recordings demonstrated that both mutant channels are insensitive to dihydropyridines (DHPs), but are blocked by diltiazem. INS-1 cells expressing Ca(v)1.3/DHPi maintained glucose- and KCl-stimulated insulin secretion in the presence of DHPs, whereas cells expressing Ca(v)1.2/DHPi demonstrated DHP resistance to only KCl-induced secretion. INS-1 cells were also stably transfected with cDNAs encoding the intracellular loop between domains II and III of either Ca(v)1.2 or Ca(v)1.3 (Ca(v)1.2/II-III or Ca(v)1.3/II-III). Glucose- and KCl-stimulated insulin secretion in Ca(v)1.2/II-III cells were not different from untransfected INS-1 cells. However, glucose-stimulated insulin secretion was completely inhibited and KCl-stimulated secretion was substantially resistant to inhibition by DHPs, but sensitive to omega-agatoxin IVA in Ca(v)1.3/II-III cells. Moreover, the L-type channel agonist FPL 64176 markedly enhanced KCl-stimulated secretion by Ca(v)1.3/II-III cells. Together, our results suggest that Ca(2+) influx via Ca(v)1.3 is preferentially coupled to glucose-stimulated insulin secretion in INS-1 cells.