Comparative Analysis of Circulating Endothelial Progenitor Cells in Age-Related Macular Degeneration Patients Using Automated Rare Cell Analysis (ARCA) and Fluorescence Activated Cell Sorting (FACS)

BackgroundPatients with age-related macular degeneration (ARMD) begin with non-neovascular (NNV) phenotypes usually associated with good vision. Approximately 20% of NNV-ARMD patients will convert to vision debilitating neovascular (NV) ARMD, but precise timing of this event is unknown. Developing a clinical test predicting impending conversion to NV-ARMD is necessary to prevent vision loss. Endothelial progenitor cells (EPCs), defined as CD34+VEGR2+ using traditional fluorescence activated cell sorting (FACS), are rare cell populations known to be elevated in patients with NV-ARMD compared to NNV-ARMD. FACS has high inter-observer variability and subjectivity when measuring rare cell populations precluding development into a diagnostic test. We hypothesized that automated rare cell analysis (ARCA), a validated and FDA-approved technology for reproducible rare cell identification, can enumerate EPCs in ARMD patients more reliably. This pilot study serves as the first step in developing methods for reproducibly predicting ARMD phenotype conversion.MethodsWe obtained peripheral venous blood samples in 23 subjects with NNV-ARMD or treatment naïve NV-ARMD. Strict criteria were used to exclude subjects with known angiogenic diseases to minimize confounding results. Blood samples were analyzed in masked fashion in two separate laboratories. EPCs were independently enumerated using ARCA and FACS within 24 hours of blood sample collection, and p<0.2 was considered indicative of a trend for this proof of concept study, while statistical significance was established at 0.05.ResultsWe measured levels of CD34+VEGFR2+ EPCs suggestive of a trend with higher values in patients with NV compared to NNV-ARMD (p = 0.17) using ARCA. Interestingly, CD34+VEGR2+ EPC analysis using FACS did not produce similar results (p = 0.94).ConclusionsCD34+VEGR2+ may have predictive value for EPC enumeration in future ARCA studies. EPC measurements in a small sample size were suggestive of a trend in ARMD using ARCA but not FACS. ARCA could be a helpful tool for developing a predictive test for ARMD phenotype conversion

Cardiopoietic cell therapy for advanced ischemic heart failure: results at 39 weeks of the prospective, randomized, double blind, sham-controlled CHART-1 clinical trial

Cardiopoietic cells, produced through cardiogenic conditioning of patients' mesenchymal stem cells, have shown preliminary efficacy. The Congestive Heart Failure Cardiopoietic Regenerative Therapy (CHART-1) trial aimed to validate cardiopoiesis-based biotherapy in a larger heart failure cohort

Stem Cells in Cardiovascular Diseases: 30,000-Foot View

Stem cell and regenerative approaches that might rejuvenate the heart have immense intuitive appeal for the public and scientific communities. Hopes were fueled by initial findings from preclinical models that suggested that easily obtained bone marrow cells might have significant reparative capabilities; however, after initial encouraging pre-clinical and early clinical findings, the realities of clinical development have placed a damper on the field. Clinical trials were often designed to detect exceptionally large treatment effects with modest patient numbers with subsequent disappointing results. First generation approaches were likely overly simplistic and relied on a relatively primitive understanding of regenerative mechanisms and capabilities. Nonetheless, the field continues to move forward and novel cell derivatives, platforms, and cell/device combinations, coupled with a better understanding of the mechanisms that lead to regenerative capabilities in more primitive models and modifications in clinical trial design suggest a brighter future

Oligonucleotide directed sequence specific recognition and alkylation of double helical DNA by triple helix formation

Chapter II: Footprinting of Oligonucleotides on Double Helical DNA using MPE•Fe(II), DNase I, and Dimethyl Sulfate Pyrimidine oligonucleotides can, when equipped with the thymidine-EDTA• Fe(II) analogue (T*), recognize and subsequently cleave double helical DNA at binding sites>15 base pairs in size. If binding affinities of unmodified oligonucleotides are to be determined under conditions relevant to those in vivo, alternate methods of detecting oligonucleotide-directed triple helix formation are required. The footprinting of short (up to 15 base pairs) triple helical regions on restriction fragment size DNA has been undertaken. Techniques for the determination of oligonucleo tide binding to double helical DNA using MPE•Fe(II), DNase I, and dimethyl sulfate have been developed. MPE•Fe(II) allows for the determination of binding site size, and has shown that oligonucleo tide binding to DNA is cation concentration, solvent, and oligonucleotide length dependent. DNase I footprinting was conducted under conditions optimal for DNase I activity (10 mM in each Mg^(+2) and Ca^(+2)), demonstrating that oligonucleotide-directed triplexes are capable of interfering with protein activity at the oligonucleotide binding site under physiological conditions, and that divalent cations can stabilize triple helix formation. Footprinting using dimethyl sulfate reveals that a single guanine 3' to the binding site becomes hyperreactive to methylation upon triplex formation. This suggests that the triplex-duplex junction involves a change in DNA conformation which is largely limited to a single base pair. DMS footprinting reveals that the oligonucleotide CT-15 (T_5(CT)_5) does not bind the terminal 2 base pairs of the binding site in plasmid PDMAG10. DMS footprinting can be used to analyze the binding of oligonucleotides to DNA under conditions not amenable to MPE•Fe(II) or DNase I activity, and to assay the kinetics of oligonucleotide binding. DMS and DNase I footprinting techniques were used to assay for the effect of oligonucleotide concentration and base composition on binding affinity. Chapter III: Oligonucleotide-Directed Triple Helix Formation using Oligonucleotides with Increased Binding Affinities The specificity offered by the triple helix motif might provide a method for the artificial repression of gene expression and viral diseases. Changes in oligonucleotide structure could be used to control oligonucleotide affinity under in vivo conditions, where temporal and spatial intracellular pH (7.0-7.4) and ionic strength are strictly regulated and cannot be altered. Substitution at position 5 of pyrimidines alters the hydrophobic driving force, base stacking, and the electronic complementarity of the Hoogsteen base pairing for triple helix formation. Incorporation of 5-substituted pyrimidines offers a method of modulating binding affinity without changing the hydrogen bonding pattern and sequence specificities of pyrimidine oligonucleotides. Replacement of 2'd eoxycytidine with 5-methyl-2'-deoxycytidine increases the oligonucleotide affinity and extends the pH range for binding. Substitution of 5-bromo-2'deoxyuridine for thymidine increases binding affinity. Oligonucleotides constructed with 2'-deoxyuridine show lower binding affinities. Pyrimidine oligonucleotides constructed from 5-iodo-2'-deoxyuridines and 5-ethynyl-2'deoxyuridines display increased binding affinities relative to thymidine, but decreased relative to 5-bromouridine containing Oligonucleotides. Substitution by ethyl, pentyl, pentynyl, 2-phenyl-ethynyl, or fluoro at the 5 position of 2'deoxyuridine or bromo at the 5 position of 2'-deoxycytidine residues results in oligonucleotides with decreased binding affinities for double helical DNA. Chapter IV: Efficient, Base-Specific Alkylation of DNA using N-Bromoacetyloligonucleotides The attachment of a non-specific diffusible cleaving functionality to a DNA binding molecule allows for the elucidation of the structural principles for DNA recognition, a technique termed affinity cleaving. Once these principles have been determined, it becomes possible to design and attach structural domains designed to carry out a desired DNA modification. The development of a thymidine derivative capable of efficient and base specific DNA modification is reported. N-bromoacetyloligonucleotides are capable of near quantitative double strand modification of double helical DNA at a single guanine position in a manner which produces ends which are ligatable with compatible ends produced by conventional restriction enzyme digestion. The products thus produced are capable of transforming bacterial cell lines. N-bromoacetyloligonucleotides modify double helical DNA with specificities great enough to produce efficient (>90%) chemical cleavage at a single site within a yeast chromosome 340 kbp in size. The acceleration obtained by tethering a reactive moiety to a DNA binding unit has been estimated. The rate of alkylation of DNA by N-bromoacetyloligonucleotides and bromoacetamide has been measured. Comparison of these ra tes indicates that an effective molarity of 2-3 M is obtained upon tethering the bromoacetyl moiety to an oligonucleotide to effect triple helix mediated DNA alkylation. The utility of the bromoacetyl moiety as a reporter group is shown in studies concerning the effect of oligonucleotide length on binding affinity and the cooperative interaction between oligonucleotides binding abutting sites is reported. </p

Sequence-specific alkylation of double-helical DNA by oligonucleotide-directed triple-helix formation

Affinity cleaving, a method that relies on the attachment of a nonspecific cleaving moiety, such as EDTA•Fe(ll), to a DNA binding molecule, facilitates the elucidation of the structural principles for DNA recognition. The determination of the sequence specificities, groove locations, and binding orientations of peptide analogues, protein-DNA binding motifs, and oligonucleotide-triple-helix motifs has provided reliable models for the sequence-specific recognition of double-helical DNA. It now becomes possible to combine these binding molecules with domains capable of base-specific and quantitative modification of DNA (Figure 1). We report the design and synthesis of an ligodeoxyribonucleotide equipped with an electrophile at the 5'-end that binds to double-helical DNA by triple-helix formation and alkylates predominantly at a single guanine base adjacent to the target DNA sequence in high yield

Triple helix formation by oligonucleotides on DNA extended to the physiological pH range

We report here that oligodeoxyribonucleotides which contain 5-bromouracil (Br^5U) and 5-methylcytosine (m^5C) bind duplex DNA at the same homopurine target sequence as their T/C analogues but with greater affinities and over an extended pH range. Oligonucleotides containing uracil (U) bind with lower affinity (Figure 1)

Review: Endothelial progenitor cells: markers of vascular reparative capacity

Assessment of the propensity for vascular events has been based on measurement of risk factors predisposing one to vascular injury. These assessments are based on the strong associations between risk factors such as hypertension, cholesterol levels, smoking, and diabetes which were first described almost a half century ago. The more recent discovery of the relationship between ongoing inflammation and clinical outcomes has led to a variety of blood-based assays which may impart additional knowledge about an individual's propensity for future cardiovascular events. Vascular health is now better represented as a balance between ongoing injury and resultant vascular repair, mediated at least in part by circulating endothelial progenitor cells. To date, one's risk for vascular events has focused exclusively on assessing propensity for vascular damage, either by assessing conventional risk factors which were initially identified over half a century ago, or more recently by assessing markers of inflammation and other circulating factors which area related to subsequent clinical events. Circulating endothelial progenitor cells play important roles in accelerating endothelialization at areas of vascular damage, and EPC enumeration is a viable strategy for assessing reparative capacity. To date, EPC numbers have been correlated with the numbers of cardiovascular risk factors, extent of coronary disease, and future cardiovascular events. Given that EPC enumeration and functional characterization represent the only assessment of the reparative side of the balance between damage and renovation, this technique may offer independent and different assessment of propensity to cardiovascular injury, greatly improving risk stratification of patients

Triple helix formation by oligonucleotides on DNA extended to the physiological pH range

We report here that oligodeoxyribonucleotides which contain 5-bromouracil (Br^5U) and 5-methylcytosine (m^5C) bind duplex DNA at the same homopurine target sequence as their T/C analogues but with greater affinities and over an extended pH range. Oligonucleotides containing uracil (U) bind with lower affinity (Figure 1)

Sequence-specific double-strand alkylation and cleavage of DNA mediated by triple-helix formation

Attachment of the nondiffusible electrophile N-bromoacetyl to the 5-position of a thymine at the 5'-end of a pyrimidine oligodeoxyribonucleotide affords sequence specific alkylation of a guanine two base pairs to the 5'-side of a local triple-helix complex in >96% yield. N-Bromoacetyloligodeoxyribonucleotides bind adjacent inverted purine tracts on double-helical DNA by triple-helix formation and alkylate single guanine positions on opposite strands at 37-degrees-C (pH 7.4). After depurination, double-strand cleavage at a single site within plasmid DNA (4 kp in size) occurs in greater than 85% yield. The resulting DNA fragments from site-specific alkylation and cleavage can be ligated with DNA fragments generated by restriction endonuclease digestion. This nonenzymatic approach which couples sequence-specific recognition with sequence-dependent cleavage affords double-strand site-specific cleavage in megabase size DNA. A yeast chromosome, 340 000 base pairs in size, was cleaved at a single site in 85-90% yield