Infliximab Limits Injury in Myocardial Infarction.

BACKGROUND: The purpose of this study was to investigate a therapeutic approach targeting the inflammatory response and consequent remodeling from ischemic myocardial injury. METHODS AND RESULTS: Coronary thrombus aspirates were collected from patients at the time of ST-segment-elevation myocardial infarction and subjected to array-based proteome analysis. Clinically indistinguishable at myocardial infarction (MI), patients were stratified into vulnerable and resilient on the basis of 1-year left ventricular ejection fraction and death. Network analysis from coronary aspirates revealed prioritization of tumor necrosis factor-α signaling in patients with worse clinical outcomes. Infliximab, a tumor necrosis factor-α inhibitor, was infused intravenously at reperfusion in a porcine MI model to assess whether infliximab-mediated immune modulation impacts post-MI injury. At 3 days after MI (n=7), infliximab infusion increased proregenerative M2 macrophages in the myocardial border zone as quantified by immunofluorescence (24.1%±23.3% in infliximab versus 9.29%±8.7% in sham; P<0.01). Concomitantly, immunoassays of coronary sinus samples quantified lower troponin I levels (41.72±7.34 pg/mL versus 58.11±10.75 pg/mL; P<0.05) and secreted protein analysis revealed upregulation of injury-modifying interleukin-2, -4, -10, -12, and -18 cytokines in the infliximab-treated cohort. At 4 weeks (n=12), infliximab treatment resulted in significant protective influence, improving left ventricular ejection fraction (53.9%±5.4% versus 36.2%±5.3%; P<0.001) and reducing scar size (8.31%±10.9% versus 17.41%±12.5%; P<0.05). CONCLUSIONS: Profiling of coronary thrombus aspirates in patients with ST-segment-elevation MI revealed highest association for tumor necrosis factor-α in injury risk. Infliximab-mediated immune modulation offers an actionable pathway to alter MI-induced inflammatory response, preserving contractility and limiting adverse structural remodeling.

Nebulized platelet-derived extracellular vesicles attenuate chronic cigarette smoke-induced murine emphysema.

Chronic obstructive pulmonary disease (COPD) is a prevalent lung disease usually resulting from cigarette smoking (CS). Cigarette smoking induces oxidative stress, which causes inflammation and alveolar epithelial cell apoptosis and represents a compelling therapeutic target for COPD. Purified human platelet-derived exosome product (PEP) is endowed with antioxidant enzymes and immunomodulatory molecules that mediate tissue repair. In this study, a murine model of CS-induced emphysema was used to determine whether nebulized PEP can influence the development of CS-induced emphysema through the mitigation of oxidative stress and inflammation in the lung. Nebulization of PEP effectively delivered the PEP vesicles into the alveolar region, with evidence of their uptake by type I and type II alveolar epithelial cells and macrophages. Lung function testing and morphometric assessment showed a significant attenuation of CS-induced emphysema in mice treated with nebulized PEP thrice weekly for 4 weeks. Whole lung immuno-oncology RNA sequencing analysis revealed that PEP suppressed several CS-induced cell injuries and inflammatory pathways. Validation of inflammatory cytokines and apoptotic protein expression on the lung tissue revealed that mice treated with PEP had significantly lower levels of S100A8/A9 expressing macrophages, higher levels of CD4+/FOXP3+ Treg cells, and reduced NF-κB activation, inflammatory cytokine production, and apoptotic proteins expression. Further validation using in vitro cell culture showed that pretreatment of alveolar epithelial cells with PEP significantly attenuated CS extract-induced apoptotic cell death. These data show that nebulization of exosomes like PEP can effectively deliver exosome cargo into the lung, mitigate CS-induced emphysema in mice, and suppress oxidative lung injury, inflammation, and apoptotic alveolar epithelial cell death.

Adeno-associated virus-9 reverses delayed gastric emptying of solids in diabetic mice.

BACKGROUND: Gastroparesis is defined by delayed gastric emptying (GE) without obstruction. Studies suggest targeting heme oxygenase-1 (HO1) may ameliorate diabetic gastroparesis. Upregulation of HO1 expression via interleukin-10 (IL-10) in the gastric muscularis propria is associated with reversal of delayed GE in diabetic NOD mice. IL-10 activates the M2 cytoprotective phenotype of macrophages and induces expression of HO1 protein. Here, we assess delivery of HO1 by recombinant adeno-associated viruses (AAVs) in diabetic mice with delayed GE. METHODS: C57BL6 diabetic delayed GE mice were injected with 1 × 1012 vg scAAV9-cre, scAAV9-GFP, or scAAV9-HO1 particles. Changes to GE were assessed weekly utilizing our [13 C]-octanoic acid breath test. Stomach tissue was collected to assess the effect of scAAV9 treatment on Kit, NOS1, and HO1 expression. KEY RESULTS: Delayed GE returned to normal within 2 weeks of treatment in 7/12 mice receiving scAAV9-cre and in 4/5 mice that received the scAAV9-GFP, whereas mice that received scAAV9-HO1 did not respond in the same manner and had GE that took significantly longer to return to normal (6/7 mice at 4-6 weeks). Kit, NOS1, and HO1 protein expression in scAAV9-GFP-treated mice with normal GE were not significantly different compared with diabetic mice with delayed GE. CONCLUSIONS AND INFERENCES: Injection of scAAV9 into diabetic C57BL6 mice produced a biological response that resulted in acceleration of GE independently of the cargo delivered by the AAV9 vector. Further research is needed to determine whether use of AAV mediated gene transduction in the gastric muscularis propria is beneficial and warranted.

Secretome signature of cardiopoietic cells echoed in rescued infarcted heart proteome.

Stem cell paracrine activity is implicated in cardiac repair. Linkage between secretome functionality and therapeutic outcome was here interrogated by systems analytics of biobanked human cardiopoietic cells, a regenerative biologic in advanced clinical trials. Protein chip array identified 155 proteins differentially secreted by cardiopoietic cells with clinical benefit, expanded into a 520 node network, collectively revealing inherent vasculogenic properties along with cardiac and smooth muscle differentiation and development. Next generation RNA sequencing, refined by pathway analysis, pinpointed miR-146 dependent regulation upstream of the decoded secretome. Intracellular and extracellular integration unmasked commonality across cardio-vasculogenic processes. Mirroring the secretome pattern, infarcted hearts benefiting from cardiopoietic cell therapy restored the disease proteome engaging cardiovascular system functions. The cardiopoietic cell secretome thus confers a therapeutic molecular imprint on recipient hearts, with response informed by predictive systems profiling.

[89Zr]Zr-DBN labeled cardiopoietic stem cells proficient for heart failure.

INTRODUCTION: Radiolabeling of stem cells with a positron emitting radioisotope represents a major advancement in regenerative biotherapy enabling non-invasive imaging. To assess the value of such an approach in a clinically relevant scenario, the tolerability and therapeutic aptitude of [89Zr]zirconium-p-isothiocyanatobenzyl-desferrioxamine ([89Zr]Zr-DBN) labeled human cardiopoietic stem cells (CPs) were evaluated in a model of ischemic heart failure. METHODS AND RESULTS: [89Zr]Zr-DBN based radiolabeling of human CPs yielded [89Zr]Zr-DBN-CPs with radioactivity yield of 0.70 ± 0.20 MBq/106 cells and excellent label stability. Compared to unlabeled cell counterparts, [89Zr]Zr-DBN-CPs maintained morphology, viability, and proliferation capacity with characteristic expression of mesodermal and pro-cardiogenic transcription factors defining the cardiopoietic phenotype. Administered in chronically infarcted murine hearts, [89Zr]Zr-DBN-CPs salvaged cardiac pump failure, documented by improved left ventricular ejection fraction not inferior to unlabeled CPs and notably superior to infarcted hearts without cell treatment. CONCLUSION: The present study establishes that [89Zr]Zr-DBN labeling does not compromise stem cell identity or efficacy in the setting of heart failure, offering a non-invasive molecular imaging platform to monitor regenerative biotherapeutics post-transplantation.

Antithrombotic effects of heme-degrading and heme-binding proteins.

In the murine venous thrombosis model induced by ligation of the inferior vena cava (IVCL), genetic deficiency of heme oxygenase-1 (HO-1) increases clot size. This study examined whether induction of HO-1 or administration of its products reduces thrombosis. Venous HO-1 upregulation by gene delivery reduced clot size, as did products of HO activity, biliverdin, and carbon monoxide. Induction of HO-1 by hemin reduced clot formation, clot size, and upregulation of plasminogen activator inhibitor-1 (PAI-1) that occurs in the IVCL model, while leaving urokinase plasminogen activator (uPA) and tissue plasminogen activator (tPA) expression unaltered. The reductive effect of hemin on clot size required HO activity. The IVCL model exhibited relatively high concentrations of heme that peaked just before maximum clot size, then declined as clot size decreased. Administration of hemin decreased heme concentration in the IVCL model. HO-2 mRNA was induced twofold in the IVCL model (vs. 40-fold HO-1 induction), but clot size was not increased in HO-2-/- mice compared with HO-2+/+ mice. Hemopexin, the major heme-binding protein, was induced in the IVCL model, and clot size was increased in hemopexin-/- mice compared with hemopexin+/+ mice. We conclude that in the IVCL model, the heme-degrading protein HO-1 and HO products inhibit thrombus formation, as does the heme-binding protein, hemopexin. The reductive effects of hemin administration require HO activity and are mediated, in part, by reducing PAI-1 upregulation in the IVCL model. We speculate that HO-1, HO, and hemopexin reduce clot size by restraining the increase in clot concentration of heme (now recognized as a procoagulant) that otherwise occurs.NEW & NOTEWORTHY This study provides conclusive evidence that two proteins, one heme-degrading and the other heme-binding, inhibit clot formation. This may serve as a new therapeutic strategy in preventing and treating venous thromboembolic disease.

M3RNA Drives Targeted Gene Delivery in Acute Myocardial Infarction.

IMPACT STATEMENT: The M3RNA (microencapsulated modified messenger RNA) platform is an approach to deliver messenger RNA (mRNA) in vivo, achieving a nonintegrating and viral-free approach to gene therapy. This technology was, in this study, tested for its utility in the myocardium, providing a unique avenue for targeted gene delivery into the freshly infarcted myocardial tissue. This study provides the evidentiary basis for the use of M3RNA in the heart through depiction of its performance in cultured cells, healthy rodent myocardium, and acutely injured porcine hearts. By testing the technology in large animal models of infarction, compatibility of M3RNA with current coronary intervention procedures was verified.

A new model of an arteriovenous fistula in chronic kidney disease in the mouse: beneficial effects of upregulated heme oxygenase-1.

The arteriovenous fistula (AVF) is the preferred hemodialysis vascular access, but it is complicated by high failure rates and attendant morbidity. This study provides the first description of a murine AVF model that recapitulates two salient features of hemodialysis AVFs, namely, anastomosis of end-vein to side-artery to create the AVF and the presence of chronic kidney disease (CKD). CKD reduced AVF blood flow, observed as early as 3 days after AVF creation, and increased neointimal hyperplasia, venous wall thickness, thrombus formation, and vasculopathic gene expression in the AVF. These adverse effects of CKD could not be ascribed to preexisting alterations in blood pressure or vascular reactivity in this CKD model. In addition to vasculopathic genes, CKD induced potentially vasoprotective genes in the AVF such as heme oxygenase-1 (HO-1) and HO-2. To determine whether prior HO-1 upregulation may protect in this model, we upregulated HO-1 by adeno-associated viral gene delivery, achieving marked venous induction of the HO-1 protein and HO activity. Such HO-1 upregulation improved AVF blood flow and decreased venous wall thickness in the AVF. Finally, we demonstrate that the administration of carbon monoxide, a product of HO, acutely increased AVF blood flow. This study thus demonstrates: 1) the feasibility of a clinically relevant murine AVF model created in the presence of CKD and involving an end-vein to side-artery anastomosis; 2) the exacerbatory effect of CKD on clinically relevant features of this model; and 3) the beneficial effects in this model conferred by HO-1 upregulation by adeno-associated viral gene delivery.

Mucosal vaccination by adenoviruses displaying reovirus sigma 1.

We developed adenovirus serotype 5 (Ad5) vectors displaying the sigma 1 protein from reovirus as mucosal vaccines. Ad5-sigma retargets to JAM-1 and sialic acid, but has 40-fold reduced gene delivery when compared to Ad5. While weaker at transduction, Ad5-sigma generates stronger T cell responses than Ad5 when used for mucosal immunization. In this work, new Ad5-fiber-sigma vectors were generated by varying the number of fiber ß-spiral shaft repeats (R) between the fiber tail and sigma. Increasing chimera length led to decreasing insertion of these proteinsAd5 virions. Ad-R3 and R14 vectors effectively targeted JAM-1 in vitro while R20 did not. When wereused to immunize mice by the intranasal route, Ad5-R3-sigma produced higher serum and vaginal antibody responses than Ad5. These data suggest optimized Ad-sigma vectors may be useful vectors for mucosal vaccination.

Induction and functional significance of the heme oxygenase system in pathological shear stress in vivo.

The present study examined the heme oxygenase (HO) system in an in vivo murine model of pathological shear stress induced by partial carotid artery ligation. In this model, along with upregulation of vasculopathic genes, HO-1 is induced in the endothelium and adventitia, whereas HO-2 is mainly upregulated in the endothelium. Within minutes of ligation, NF-κB, a transcription factor that upregulates vasculopathic genes and HO-1, is activated. Failure to express either HO-1 or HO-2 exaggerates the reduction in carotid blood flow and exacerbates vascular injury. After artery ligation, comparable induction of HO-2 occurred in HO-1(+/+) and HO-1(-/-) mice, whereas HO-1 induction was exaggerated in HO-2(-/-) mice compared with HO-2(+/+) mice. Upregulation of HO-1 by an adeno-associated viral vector increased vascular HO-1 expression and HO activity and augmented blood flow in both ligated and contralateral carotid arteries. Acute inhibition of HO activity decreased flow in the ligated carotid artery, whereas a product of HO, carbon monoxide (CO), delivered by CO-releasing molecule-3, increased carotid blood flow. In conclusion, in the partial carotid artery ligation model of pathological shear stress, this study provides the first demonstration of 1) upregulation and vasoprotective effects of HO-1 and HO-2 and the vasorelaxant effects of CO as well as 2) vascular upregulation of HO-1 in vivo by an adeno-associated viral vector that is attended by a salutary vascular response. Induction of HO-1 may reside in NF-κB activation, and, along with induced HO-2, such upregulation of HO-1 provides a countervailing vasoprotective response in pathological shear stress in vivo.

Effects of adeno-associated virus serotype and tissue-specific expression on circulating biomarkers of propionic acidemia.

Propionic acidemia (PA) is an autosomal recessive inborn error of metabolism caused by deficiency of propionyl-CoA carboxylase (PCC). This enzyme is composed of six PCCA and six PCCB subunits and mediates a critical step in catabolism of odd chain fatty acids and certain amino acids. Current treatment options for PA are limited to stringent dietary restriction of protein consumption and some patients undergo elective liver transplantation. We previously generated a hypomorphic model of PA, designated Pcca(-/-)(A138T), with 2% of wild-type enzyme activity that mimics many aspects of the human disease. In this study, we used the differing tissue tropisms of adeno-associated virus (AAV) to probe the ability of liver or muscle-directed gene therapy to treat systemic aspects of this disease that affects many cell types. Systemic therapy with muscle-biased AAV1, liver-biased AAV8, and broadly tropic AAVrh10 mediated significant biochemical corrections in circulating propionylcarnitine (C3) and methyl citrate by all vectors. The innate tissue bias of AAV1 and AAV8 gene expression was made more specific by the use of muscle-specific muscle creatine kinase (specifically MCK6) and hepatocyte-specific transthyretin (TTR) promoters, respectively. Under these targeted conditions, both vectors mediated significant long-term correction of circulating metabolites, demonstrating that correction of muscle and likely other tissue types in addition to liver is necessary to fully correct pathology caused by PA. Liver-specific AAV8-TTR-PCCA mediated better correction than AAV1-MCK-PCCA. These data suggest that targeted gene therapy may be a viable alternative to liver transplantation for PA. They also demonstrate the effects of tissue-specific and broad gene therapy on a cell autonomous systemic genetic disease.

Generation of a hypomorphic model of propionic acidemia amenable to gene therapy testing.

Propionic acidemia (PA) is a recessive genetic disease that results in an inability to metabolize certain amino acids and odd-chain fatty acids. Current treatment involves restricting consumption of these substrates or liver transplantation. Deletion of the Pcca gene in mice mimics the most severe forms of the human disease. Pcca(-) mice die within 36 hours of birth, making it difficult to test intravenous systemic therapies in them. We generated an adult hypomorphic model of PA in Pcca(-) mice using a transgene bearing an A138T mutant of the human PCCA protein. Pcca(-/-)(A138T) mice have 2% of wild-type PCC activity, survive to adulthood, and have elevations in propionyl-carnitine, methylcitrate, glycine, alanine, lysine, ammonia, and markers associated with cardiomyopathy similar to those in patients with PA. This adult model allowed gene therapy testing by intravenous injection with adenovirus serotype 5 (Ad5) and adeno-associated virus 2/8 (AAV8) vectors. Ad5-mediated more rapid increases in PCCA protein and propionyl-CoA carboxylase (PCC) activity in the liver than AAV8 and both vectors reduced propionylcarnitine and methylcitrate levels. Phenotypic correction was transient with first generation Ad whereas AAV8-mediated long-lasting effects. These data suggest that this PA model may be a useful platform for optimizing systemic intravenous therapies for PA.

Circulating antibodies and macrophages as modulators of adenovirus pharmacology.

Adenovirus serotype 5 (Ad5) naturally infects the liver after intravenous injection, making it a candidate for hepatocyte-directed gene transfer. While Ad5 can be efficient, most of the dose is destroyed by liver Kupffer cells before it can reach hepatocytes. In contrast, Ad5 bearing the hexon from Ad6 (Ad5/6) evades Kupffer cells. While Ad5/6 dramatically increases hepatocyte transduction in BALB/c mice, it has surprisingly little effect on C57BL/6 mice. To determine the source of this strain-specific difference, the roles of Kupffer cells, liver sinusoidal endothelial cells (LSECs), hepatocytes, scavenger receptors, clotting factors, and immunoglobulins were analyzed. The numbers of Kupffer cells and LSECs, the level of clotting factor X, and hepatocyte infectibility did not differ between different strains of mice. In contrast, high levels of immunoglobulins correlated negatively with Ad5 liver transduction in different mouse strains. Removal of immunoglobulins by use of Rag-deficient mice restored Ad5 transduction to maximal levels. Removal of Kupffer cells by predosing or by testing in colony-stimulating factor knockout mice restored Ad5 transduction in the presence of immunoglobulins. Partial reconstitution of IgM in Rag mice resulted in significant reductions in liver transduction by Ad5 but not by Ad5/6. These data suggest a role for IgM-mediated clearance of Ad5 via Kupffer cells and may explain the mechanism by which Ad5/6 evades these cells. These mechanisms may play a vital role in Ad pharmacology in animals and in humans.

A vector-host system to fingerprint virus tropism.

Reporter genes are important tools for assessing vector pharmacology in vivo. Although useful, current systems are limited by (1) the need to generate a new vector for each different reporter, (2) the inability to package reporter genes in small vectors, and (3) variations in reporter gene feedback due to variations in cell-to-cell vector copy number. To circumvent these problems, we have used Cre recombinase as a "cat's paw" to activate reporter genes embedded in transgenic mice. The small Cre gene was introduced into self-complementary adeno-associated viral (scAAV) vectors with limited packaging capacity. Injection of scAAV-Cre vectors into mice with loxP-inactivated luciferase enabled in vivo imaging distributions comparable to the signal observed after AAV-luciferase injection. When injected into mT/mG mice, AAV-Cre converted ubiquitous expression of red fluorescent protein (RFP) to green fluorescent protein (GFP) expression only where the vectors transduced cells. Injection into F(1) hybrid luciferase and mT/mG mice enabled simultaneous three-reporter tracking. This system was able to discriminate cell-specific transduction in all organs tested, with particular usefulness for detecting AAV serotype-specific transduction in the liver, kidney, and muscle. Given that F(1) mice bear exactly one copy of luciferase and one copy of RFP-GFP, each reporter gene is either "on" or "off" in a cell. The Cre system therefore provides a unique quantum method to quantify vector delivery that can be applied when vector capacity is limited.

Systemic delivery of therapeutic viruses.

The treatment of certain diseases will require the systemic delivery of therapeutic genes or viruses. In most cases, intravascular injection is the best delivery method to achieve the systemic distribution of viruses and to enable these agents to reach distant therapeutic sites. However, viruses administered by intravascular injection encounter overlapping barriers that impede their ability to reach their targets, including interactions with blood cells, blood factors and endothelial cells, loss to hepatocytes and macrophages, and destruction by innate and adaptive immune responses. In this review, recent advances in the understanding of the mechanisms determining virus tropism following systemic administration and the pharmacology of therapeutic viruses are described. Adenoviruses are used as a paradigm of these interactions, and factors affecting their therapeutic efficacy and side effects are discussed, as well as how the barriers that impede their ability to reach their targets translate to other therapeutic viruses.

Converting tumor-specific markers into reporters of oncolytic virus infection.

Preferential killing of transformed cells, while keeping normal cells and organs unharmed, is the main goal of cancer gene therapy. Genetically engineered trackable markers and imaging reporters enable noninvasive monitoring of transduction efficiency and pharmacokinetics of anticancer virotherapeutics. However, none of these reporters can differentiate between infection in the targeted tumors and that in the normal tissue. Thus, we constructed oncolytic measles virus (MV) armed with a human light immunoglobulin chain reporter gene for the treatment of multiple myeloma (MM). Excessive production of monoclonal immunoglobulin is a key characteristic and marker for diagnostics of MM. Once expressed in infected target cells, vector-encoded lambda protein recombines with myeloma IgG-kappa immunoglobulin creating a unique IgG-kappa/lambda. A modified immunoassay technique allows precise quantification of converted marker molecules. Only antibody producing cells were able to assemble this chimeric immunoglobulin molecule, whereas other cells secreted only free lambda light chain. Human myeloma xenografts inoculated with lambda chain expressing MV secreted converted IgG-kappa/lambda in the plasma of tumor bearing animals and elevated reporter levels correlated with response to the therapy. This is the first report of a gene therapy vector engineered to discriminate between infection in malignant and normal cells by molecular modification of a tumor-specific protein.

Comparison of TRY and the closely related At1g01380 gene in controlling Arabidopsis trichome patterning.

A screen of activation-tagged Arabidopsis lines resulted in the identification of At1g01380, which encodes a small R3 single repeat MYB gene, as a negative regulator of trichome initiation. Plants that overexpress this gene have fewer trichomes. The gene is closely related to the previously identified negative regulator TRY, and has a similar pattern of expression as TRY in developing leaves. As previously shown for TRY, At1g01380 protein can inhibit the interaction between the positive trichome regulators GL1 and GL3, and likely limits trichome initiation via this inhibition. While TRY and At1g01380 are closely related, they are not completely functionally equivalent. When placed under the transcriptional control of the TRY promoter, At1g01380 can only partially rescue the try mutant. Interestingly, Atg01380 is highly expressed in gl3-sst trichomes, while TRY expression is greatly reduced. The mutation in gl3-sst causes a reduced interaction between the GL1 and GL3 proteins and results in fewer leaf trichomes that develop in clusters. The differential expression of TRY and At1g01380 in this mutant can be used to explain how its altered trichome pattern in gl3-sst [corrected] is generated.

Cis-acting elements stimulating kinetoplastid guide RNA-directed editing.

The coding sequence of several mitochondrial mRNAs of the kinetoplastid protozoa is created through the insertion and deletion of specific uridylates. The editing reactions are required to be highly specific in order to ensure that functional open reading frames are created in edited mRNAs and that potentially deleterious modification of normally nonedited sequence does not occur. Selection-amplification and mutagenesis were previously used to identify the optimal sequence requirements for in vitro editing. There is, however, a minority of natural editing sites with suboptimal sequence. Several cis-acting elements, obtained from an in vitro selection, are described here that are able to compensate for a suboptimal editing site. An A + U sequence element within the 5'-untranslated region of cytochrome b mRNA from Leishmania tarentolae is also demonstrated to function as a cis-acting guide RNA and is postulated to compensate for a suboptimal editing site in vivo. Two proteins within an enriched editing extract are UV-cross-linked to two different in vitro selected editing substrates more efficiently than poorly edited RNAs. The results suggest that these proteins contribute to the specificity of the editing reaction.

A contradictory GLABRA3 allele helps define gene interactions controlling trichome development in Arabidopsis.

Previously characterized Arabidopsis gl3 mutants have trichomes that are smaller, less branched and undergo fewer rounds of endoreplication than wild-type trichomes. A new gl3 mutant, called gl3-sst, has oddly shaped trichomes that over expand during early development, undergo more endoreduplication and that have a striking nuclear morphology. The mutant nuclei consist of many interconnected lobes; however, only a single set of polytene-like chromosomes reside in the mutant nuclei. The predicted gl3-sst polypeptide has a Leu to Phe substitution (codon 78) within a region responsible for protein-protein interaction. Yeast interaction assays comparing GL3 with gl3-sst proteins show that the mutant protein interaction with GL1 and TTG1 is decreased by 75% and 50%, respectively, but there is no difference in its interaction with TRY. Furthermore, TRY has the ability to prevent the GL1 GL3 interaction and the GL1 gl3-sst interaction is even more sensitive to TRY. Analysis of plants expressing functional GFP-tagged versions of GL1, GL3 and TRY show that the proteins are localized in trichome nuclei. These results have been used to model trichome initiation in terms of protein interactions and threshold levels of activator complex.