Heparin, Growth Factors and Rosacea

Knock-out Mice and FGF Receptor Inhibitors Mimic Rosacea
Heparin Nanofibers Loaded with VEGF and FGF Mimic Stem Cells

In previous articles, I have emphasized the mediation of extracellular signaling by heparan sulfate proteoglycans (HSPGs, polysaccharides attached to proteins) and heparin (HS fragments, oligosaccharides) and the sensitivity of HSPG expression and HS degradation by inflammation.  I return to that subject, spurred on by reading two articles that together show both the significance of heparin-mediated growth factors in general and in the specific case of symptom development in rosacea.

FGF Receptor Inhibitors Cause Symptoms Like Rosacea
Fibroblast growth factors stimulate the development of cancers, and antibodies against FGF receptors block cancer growth (see ref.)  FGF receptor inhibiting antibodies are now being used to stop cancers.  Unfortunately,  FGFR antibodies (e.g. cetuximab, panitumumab) also cause symptoms in the skin (telangiectasia, acneiform eruption) similar to the facial inflammation of rosacea.  Similarly, in knock-out mice, that lack the ability to produce FGFR, there are related symptoms.  It appears that lack of some FGF signaling may produce the symptoms of visible blood vessels and pus-filled (though lacking bacteria) follicles of rosacea.

FGF Mediated by HSPG
FGF binds to the heparan sulfate of membrane bound HSPG in pairs and these FGF dimer/heparan sulfate complexes activate a pair of FGF receptors.  The result is activation of protein phosphorylation activity (tyrosine kinase) and normal skin development.  HSPG synthesis is modified by inflammation and heparanase activity is increased.  This suggests that inflammation will decrease FGF signaling and could lead to symptoms of rosacea.

Growth Factors (VEGF, FGF) Bind to Heparin Nanofibers that Mimic Stem Cells
Stem cells produce lots of different growth factors and when stem cells are introduced into damaged cardiovascular tissue, more healing results (see ref.)  To determine if the growth factors produced by the transplanted stem cells was sufficient for the improved healing, fibers made of heparin were dipped into stem cell cultures and the resulting growth factor-coated fibers were injected into damaged tissue.  The heparin-binding growth factors were just as effective at enhancing healing as were the stem cells in previous experiments.  This demonstrated that heparin-binding growth factors were the key to normal repair/revascularization and function.

Rosacea Results from Inflammation and Aberrant Vascularization
Rosacea is poorly understood and is probably numerous diseases that have related symptoms and complex development.  As I indicated in previous articles, neurotransmitters from stimulated facial nerves, enzymes (kallikrein) and cytokines from intestinal interactions with gut flora, mast cell products (heparin, protease) and modified antimicrobial peptides (cathelicidins), as well as cryptic bacteria in facial tissues, may all be involved.  Inflammation in the skin of the face and in the intestines is involved.  Vitamin D, omega-3 fatty acids and anti-oxidants have a variety of responses (sometimes paradoxical) that differ from individual to individual and at different stages in the development of the disease.  Facial inflammation leads to abnormal development of blood vessels (telangiectasia) and in accumulation of lymphocytes and neutrophils (papulopustular rosacea).

Facial Inflammation May Depress HSPG Production and Disrupt FGF Function
One of the key ramifications of persistent facial inflammation may be the depletion of of HSPGs that normally coat cells.  HSPGs are continually produced, reabsorbed and degraded.  The half-life for HSPGs, even those that surround the cells that produce cartilage in connective tissue, is six hours.  HSPGs are also the source of heparin, that is produced as a counter ion bound to histamine and proteases in the secretory granules released by activated mast cells.  Thus, inflammation-based depression of HSPG production, which is also accompanied by heparanase activation, will remove the HSPG coating of cells.  This HSPG coating is needed for normal growth factor function.  Lack of an HSPG matrix on the surface of cells will also result in the migration of growth factors away from where they are normally functional and into adjacent tissue where they may stimulate aberrant development of blood vessels.  This may explain telangiectasia.

Is Topical Heparin a Rosacea Treatment?
Topical heparin does penetrate the skin.  It would appear to be a logical treatment, if HSPG depletion is contributing to symptom development in rosacea.  The length of the heparin fragments may be important.  I am unaware if anyone has tried the heparin lotions that are available for treatment of wounds to minimize scarring, on rosacea.  Heparin may be useful in combination with vitamin D3 and remediation of gut flora in a general scheme to treat rosacea.

refs:
Segaert S, Van Cutsem E.  Clinical signs, pathophysiology and management of skin toxicity during therapy with epidermal growth factor receptor inhibitors.  Ann Oncol. 2005 Sep;16(9):1425-33. Epub 2005 Jul 12.

Webber MJ, Han X, Prasanna Murthy SN, Rajangam K, Stupp SI, Lomasney JW.  Capturing the stem cell paracrine effect using heparin-presenting nanofibres to treat cardiovascular diseases.  J Tissue Eng Regen Med. 2010 Mar 10. [Epub ahead of print]

Suffering from Inflammation?

How do you know if your symptoms result from inflammation?

My interest is the molecular basis of inflammation, how inflammation is triggered and how inflammation contributes to numerous diseases. I try to expose the inflammatory underpinnings of various diseases by initially linking a disease to inflammation and then unraveling the molecular events that lead to and make up the disease.

How Do I Link a Disease to Inflammation?

My first task is to check the biomedical literature to see if there are research articles that support anti-inflammatory interventions that prevent or limit the disease. I just do a PubMed search the disease name plus anti-inflammatory treatments, e.g. omega-3 fish oils, vitamin D, NSAIDs, etc. It is also possible to see if a disease, such as diabetes, that produces chronic inflammation is a risk factor for the new disease being examined. It is shocking to me that omega-3 fish oils (EPA/DHA) or even flax seed oil, have been found to be effective treatments for numerous diseases that range from allergies, arthritis, inflammatory bowel diseases, depression and even septic shock and multiple organ failure. Aspirin has been used to treat infertility and post partum depression, and at high levels to treat cancer.

Dietary Suppression as Prima Fascia Evidence of Inflammatory Cause

If I find that omega-3 oils have been used successfully to treat a disease, then I attempt to link inflammation to the molecular events that initiate the disease. The biomedical literature is of minimal help here. [Biomedical research is usually limited to assessing the impact of drugs on the symptoms of diseases, so the biomedical literature typically does not provide information on the cause of diseases or ways to cure diseases. Causes and cures do not receive research funding.] I have to learn the basic workings of the organs involved and the alterations of function associated with the disease. I have also found by long experience, that major molecular components are systematically missing from typical explanations of function.

Heparan sulfate/heparin Is Missing in Action

Heparan sulfate proteoglycans (HSPGs) dominate the extracellular environment and yet they are systematically excluded from biomedical research. On this blog, I have provided dozens of examples of the essential role played by HSPGs and disruption of these roles by heparin. The majority of cytokines, growth factors, clotting events, complement cascades and even lipid transport (LDL) act via HSPGs. Leaking of proteins into the urine, across the intestines or the blood brain barrier is controlled by HSPGs, is reduced by inflammation and can be partially repaired by heparin. Autoimmune and allergic diseases are initiated by disruptions in HSPG metabolism. Viral and bacterial pathogens bind to human cells via HSPGs. Cancer cells reduce their HSPGs and start to secrete heparanase in order to metastasize. Mast cells secrete heparin! HSPGs and heparin are major players in tissue function and yet the major cell biology text book does not even discuss them. HSPGs are not mentioned in medical school training even though heparin is the most commonly administered drug.

One of the insights that I bring to my conceptualization of diseases is the role of heparan/heparin in cellular physiology. It explains a lot.

Check for Inflammatory Symptoms by Trying the Anti-Inflammatory Diet

If your symptoms are due to inflammation, there is an easy way to find out. Since diet is the biggest source of inflammation and most of the cells of the immune system congregate in your intestines, it makes sense to check to see your health problems are rooted in inflammation by making simple changes in your diet. Since this is just a test, don’t worry about whether or not this is diet for the rest of your life. Just stick to it for a week and see if it changes your life.

The Basic Anti-Inflammatory Diet and Lifestyle Guidelines are here.

(Vitamin D and omega-3 fish oil amounts are minimal levels. More severe examples of inflammation will require higher levels. Vitamin D up to 10,000 IU per day has been found safe. Some individuals require up to 12 fish oil capsule per day to experience relief from symptoms. Increases should be gradual over weeks of time.)

Try it for a week and let me know if your symptoms disappear. The prevalence of diet-based inflammation, makes me confident that you will be glad that you tried these simple, healthy changes. For immediate relief of pain, see my articles on capsaicin, castor oil and menthol/Vicks.

This is not medical advice and is used only in appropriate support of primary medical care.

Extreme Flu Remedies

Experimental Therapies for ARDS, Cytokine Storms

Do not do this at home. There are doctors and hospitals. Use them.

....But, if a doctor emailed me pleading for any ideas that I had to save a bunch of patients suffering from acute respiratory distress syndrome (ARDS) from Tamiflu-resistant H1N1, my first response would be to suggest therapies designed for ARDS from other origins, e.g. burns, septicemia, etc.

Cytokine Storms Are Out of Control
When too much tissue is injured, the local, molecular communication that normally occurs just between cells, spills into the blood stream and becomes potentially lethal. That is what happens in anaphylactic shock. It is also what happens in cytokine storms, where inflammatory cytokines that are normally short-lived and processed locally to progress into recovery, erupt into the blood stream and impact distant organs.

Major disruption of body function by aggressive blood infections or burns over most of the body, will be lethal without heroic medical interventions. These are injuries beyond the evolved adaptations of mammals.  Until recently there were no survivors.

Influenza has been around for a long time. Humans, other mammals and birds get the flu and get over it. Many body cells become infected, antibodies specific to the virus are produced within about a week, the infected cells are killed, the virus is digested and life goes on.

People die from the flu, because an opportunistic pathogen causes a lethal secondary infection, or the body over-reacts and damages itself in attempts to attack its own infected cells. This is a cytokine storm.

Silence the Storms
Cytokine storms can be weathered by blocking the signaling system. Cytokines are just small proteins that are complementary in shape to corresponding protein receptors that penetrate through the surface membranes of cells throughout the body. Binding of cytokine to receptor changes the shape of the receptor and transmits a signal into the cytoplasm of the receptive cell. This turns on aggressive behavior of immune cells and triggers more inflammatory signaling in other cells. This causes fever, malaise, etc.

...But, I was the one the doctor is pleading with to save the people. And I know that there is more to cytokine signaling than just cytokines and receptors. There are also heparan sulfate proteoglycans (HSPGs). Cytokines are not supposed to be broadcast throughout the body. Cytokines function in the space between cells, the extracellular matrix. Polysaccharides attached to membrane proteins, HSPGs, are secreted at one end of the cells, sweep across the surface and are taken back up at the other end. Cytokines have heparan-binding domains and so they stick to the heparan and are swept along. Cytokines can move from one cell to another as the sweeping HSPGs of adjacent cells come in contact.

HSPGs Mediate Cytokine Signaling
The critical point here is that cytokines bind to their receptors with the heparan between -- the cytokine and receptor are like two halves of a bun and the hot dog is the heparan. In fact the heparan bridges two cytokine/receptor complexes to make an active, signaling pentamer.

Heparin Can Block Cytokine Signaling
Heparin is a fragment of heparan sulfate produced by enzymatic degradation of HSPG. Commercial heparin, used to block blood clotting, is obtained from the mast cells of lungs and intestines of hogs and cattle. The mast cells release heparin and histamine in response to parasites or pollen. Since heparin is a short version of heparan sulfate, it can block the formation of active cytokine/receptor complexes.

Heparin is used in a mist to treat the lungs of burn patients. It is also injected into some infertility patients to suppress inflammation that is inhibiting implantation and gestation. It is also effective in treatment of autoimmune inflammation in Crohn’s disease. I think it should be tested as a therapy for H1N1 cytokine storms. It may be useful in nebulizing mists and oral treatment of intestines.

Berberine Binds to HSPG
Berberine is a phytochemical from Barberry traditionally used in the treatment of intestinal infections and arthritis. It also binds to heparan sulfate to form fluorescent complexes visible in microscopy. Berberine-treated mast cells glow brightly. Heparan sulfate can also be detected in Alzheimer’s plaque, atherosclerotic plaque and prion complexes. Because berberine binds to heparan sulfate, it should also disrupt cytokine signaling. It has been used successfully in treatment of septicemic ARDS.

Curcumin Blocks NFkB
One of the most potent chemicals that blocks inflammatory signaling via the inflammatory transcription factor, NFkB, is curcumin. Curcumin is a major component of the spice turmeric. Oral curcumin can be enhanced by co-administration of black pepper, because the piperine in pepper inhibits intestinal inactivation.

Anti-Inflammatory Diet
Of course, I would also recommend vigorous implementation of an anti-inflammatory diet and lifestyle to support any medical treatment.

Stem Cells Using HSPG Uptake of Recombinant Transcription Factors

Stem Cells from Adult Cells using Transcription Factor Genes
Stem cells have been produced from adult cells using transformation with genes for transcription factors. The problem with this approach was that the embryonic transcription factors had a tendency to promote cancer-like proliferation. What was needed was a temporary push toward embryonic gene expression by temporarily introducing a dose of embryonic transcription factors to dominate gene expression long enough to convert adult, differentiated cells into pluripotent stem cells.

Transcription Factors Synthesized by Recombinant Bacteria
The technical solution was tested and successful results were announced in a prior to publication paper in the journal Cell Stem Cell. Four transcription factors successfully used in prior experiments to induce stem cell transformation were synthesized using recombinant bacteria. The problem was getting the proteins into skin cells that were already growing in cell culture.

Protein Uptake via Triplets of Basic Amino Acids (Heparin-Binding Domains, NLS)
Transcription factors bind to DNA via basic amino acids and many of those basic amino acids are parts of the nuclear localization signals (NLS, quartet or two neighboring pairs of basic amino acids) that bind to importin and transport transcription factors from the cytoplasm to the nucleus.

HSPG Circulation Should Take in Transcription Factors
By inspection, I have demonstrated that proteins observed to be taken up by cells, without specific receptors, e.g. HIV-TAT, lactoferrin, heparanase, allergens, autoantigens, have triplets (or neighboring pairs) or basic amino acids, and this uptake is inhibited by heparin. One would expect that transcription factors would be naturally taken into cells by HSPG circulation. Just adding recombinant transcription factors to cultured skin cells should transform them into stem cells. I don’t believe that this was tested. Instead, more powerful heparin-binding domains were added.

Poly Arginine was used for Uptake of Transcription Factors
The investigators ensured a high efficiency of uptake by adding potent poly arginine sequences to the ends of the transcription factors and synthesized them in recombinant bacteria. The recombinant, arg-tailed transcription factors were taken up by the cultured skin cells and changed the pattern of gene expression in the skin cells. The cultured cells reverted to embryonic patterns of gene expression of pluripotent stem cells. The recombinant proteins were eventually metabolized, but the stem cells had been stably transformed.

reference:
Zhou, H. et al., Generation of Induced Pluripotent Stem Cells Using Recombinant Proteins, Cell Stem Cell (2009), ahead of publication 04.005

ASP, C3 and Lipid Metabolism

Each gene codes for a protein with multiple molecular actions.

I was recently reading Nigee’s Diet and Nutrition Blog and was reminded of the interesting complexity of the molecular underpinnings of physiology. Nige was discussing fat metabolism and indicated that a protein called Acylation Stimulation Protein (ASP), increased triglyceride synthesis and that ASP levels in the blood increased after a lipid-rich meal.

Since I could recall nothing about ASP, I started to examine the gene that codes for ASP and soon found that it was called C3, as in complement C3. So the lipid manipulating function is part of the repertoire of the C3 protein that also regulates the innate immune system and is sometimes referred to as an anaphylaxin, in tribute to its ability to stimulate acute inflammatory responses (anaphylactic shock).

It is not unusual for a gene to code for multiple proteins with mix-and-match domains, as a result of alternative splicing events at the mRNA processing level. That is how different classes of immunoglobulins, IgG vs. IgE, can have the same variable regions for antigen binding, but different conserved regions for binding to other parts of the immune system. In the case of complement factor C3, a single protein interacts with dozens of different proteins and is involved many different cell and tissue functions.

The versatility of C3 is partly a result of having multiple pairs of basic amino acids that can participate in binding to heparan sulfate proteoglycans (HSPGs) on the surface of cells. C3 and all of the other proteins of the complement cascade have heparin-binding domains. Thus, the complement proteins are all bound together on the surface of cells by the strands of heparan sulfate. This brings the proteins all together for interactions and during an immune assault the complement components are ultimately assembled into tunnels that breach invading cells.

The ASP-C3 story shows that the complement system is also wedded to lipid metabolism. Another juxtaposition of lipids with immune function is the lipid deposition in athersclerosis and also the association between the ApoE4 type of lipoprotein and susceptibility to Alzheimer’s disease. Clearly, lipid metabolism is intimately associated with degenerative and autoimmune diseases, as well as cancer.

Cartilage as Rejuvenation

I have studied cartilage secreting (chondrocytes) for the last few years. Chondrocytes are normally derived by differentiation of mesenchymal stem cells (MSCs) that grow in the bone marrow. MSCs can differentiate to produce bone secreting cells (osteoblasts), muscle cells (myoctes), fat cells (adipocytes) and insulin-secreting cells (beta-pancreatic islet cells.) I chose to study a rat chondrosarcoma (RCS) cell line, because this is a type of cell that shares the properties of many other important cells and it will continue to grow in cell culture. Thus, I can dilute some RCS cells in a solution with all of the nutrients required for growth and the cells will stick to the surface of the plastic dishes that I use, grow and differentiate. If you stain the cultures for cartilage, you get the following micrograph.

At first the cells stretch out and move about the surface of the dish. Then they become progressively less adhesive to the surface and more spherical as they start to produce and secrete the polysaccharides (glycosaminoglycans, such as chondroitin sulfate and heparan sulfate) and proteins (collagen) of cartilage. Finally they produce thick layers of cells that are separated and embedded in cartilage. After a little more than a week in culture, the cells are moving through the cartilage matrix by enzymatically degrading the cartilage ahead of them and secreting new cartilage in their wake. The cells that eat their way to the dish surface separate the cartilage layer from the dish and the colonies of cells begin to slough off from the dish surface. Normal chondrocytes would eventually stop dividing under culture conditions, but the cancer line that I use continues to grow quite happily and can be diluted and plated continuously. Chondrocytes in cartilage live in cavities within the cartilage and are surrounded by heparan sulfate attached to proteins of their cell membranes, i.e. heparan sulfate proteoglycans.

Cut cartilage will grow back together as the chondrocytes mine, secrete and gradually knit the two surfaces together with strands of new cartilage. Movement through and renewal of cartilage, e.g. in the connective tissue of skin, is restricted if the collagen fibers that are assembled outside of the secreting cells are cross-linked. This is why sunbathing ages skin. High fructose corn syrup also accelerates cross-linking. This cross-linking is also what makes meat less tender. The cross-linking and toughness can be measured by inserting fluorescence-measuring probes into meat, because the protein cross-links fluoresce in UV light.

A consequence of the development of chondrocytes on the ends of bones, is that the nutrients for the cells change. Initially the chondrocytes enjoy the abundant glucose and oxygen of the blood stream and gradually they are remove further from blood vessels. (Note that cartilage actively inhibits vascularization, so there are no blood vessels in cartilage. This lack of blood vessels and associated enhanced risk of disfiguring infection, is a reason to discourage piercings that involve cartilage.) Chondrocytes snug in their little cartilage cavities no longer eat sugar or breath oxygen, they dine on cartilage and ferment.

What happens if you expose mature chondrocytes to a new source of rich nutrients? I think that the answer is rejuvenation. Quite literally, the chondrocytes regress and return to the lifestyle of their youth. Instead of producing mature, weight-bearing, dense cartilage, these rejuvenated cells start to produce the weaker matrix of their youth. This weak cartilage is readily damaged by abrasion and is not suitable for joint surfaces. This is one of the consequences of arthritis. Inflammation of cartilage brings rejuvenating, damaging nutrients to chondrocytes. The mechanical damage leads to destructive cycles of further inflammation.

Chondrocytes in cartilage also respond to mechanical stress and this stress maintains their maturity. Persistent weight bearing at the same orientation leads to bone production. Thus, after joint injury it is important to use the correct regimen of rehabilitation to maintain mobility of the joint and mineralization of the bone.

Mast Cell Heparin

Mast cells are sentinels in tissues. They respond to invading pathogens by releasing their stored histamine, enzymes and heparin. The heparin modifies the activity of enzymes and cytokines.

What are mast cells and why are they loaded with heparin (left)? Mast cells start in the bone marrow, like many other components of the immune system. They then move into the blood stream and offload in most of the tissues that typically encounter pathogens and parasites. Thus, the typical commercial source of the mast cell-produced heparin is pig intestines or cow lungs, i.e. since heparin is made and stored in mast cells and mast cells are abundant in lungs and intestines, those are the sources of crude heparin. Proteins bound to the crude heparin are removed as the heparin is cleaned up to be used as an anti-clotting drug.

Mast cells are sentinels near the surface of mucus membranes that line the airways of the lungs and the digestive tract. Diseases of the lungs and intestines, e.g. asthma and inflammatory bowel disease, that have an inflammatory and/or autoimmune component yield high levels of mast cells in the affected tissues. Pathogens or parasites coming in contact with mast cells trigger the sudden release of vesicles full of histamine, enzymes and heparin.

Heparin stored in vesicles in mast cells can also be readily visualized by staining the mast cells in microscope sections using the fluorescent dye berberine (left). Berberine binds quite specifically to heparin and is also used in herbal medicine as a treatment for many inflammatory diseases, such as arthritis. It would be very interesting to know whether berberine has any effect on asthma.

Mast cells display a variety of receptor proteins on their surfaces. Protein receptors work by binding target molecules, ligands, changing their shapes and transmitting a signal through the cytoplasm. A key aspect of the signal transmission is the requirement for the ligand binding to bring together receptors in pairs. The pairing of receptors during ligand binding is facilitated by the binding of heparin to both ligands and receptors. Two ligands, e.g. cytokine peptides, such as TNF, can bind to adjacent sites on a heparin molecule and this pair can then bind to two receptors brought together on the surface of a cell. The receptors bind to the ligand and to the heparin. Some ligands will bind to their receptors without heparin, but the presence of heparin greatly accelerates and intensifies the reactions.

Heparin is synthesized in the vesicles of mast cells and binds to enzymes, e.g. tryptase, also present in the vesicles. The tryptase enzyme proteins form tetramers with heparin wrapped around the edge (left, edge view showing one pair of tryptase proteins with heparin bound diagonally to blue heparin-binding domains; other pair of tryptase proteins is hidden).

Interestingly the active site for each tryptase in the tetramer faces a hole where the four proteins come together. Thus the tetramer can degrade small peptides, but large proteins cannot get access to the blocked active sites. Monomers change shape and are no longer active.

Activated mast cells release their vesicle contents with some enzymes active and their bound heparin is replaced by the heparan sulfate attached to adjacent cells. Other enzymes are initially inactive bound to heparin and are activated by dissociation of the heparin once they are released from the vesicles. In both cases some of the heparin is released from the mast cells into the surrounding tissue. The free heparin can bind to cytokines released from other cells and the combined pairs of cytokines bound to heparin can in turn bind to appropriate receptors on other cells. The abundance of heparan sulfate bound to other cells will determine whether additional heparin is required for receptor responses from particular cytokines. Cells with abundant heparan sulfates will sweep heparin binding ligands toward receptors aggregated in lipid rafts, as the heparan sulfate proteoglycans are internalized for recycling.

Mast cells can be activated by allergens, because of IgE receptors. IgEs are antibodies that trigger allergic responses. The IgEs produced by antibody producing B lymphocytes circulate in the blood serum and bind to mast cell receptor proteins. Allergen molecules bind to the IgE-receptor complexes, trigger the activation of the mast cells and release histamine. The histamine binds to receptors on other cells and produces the symptoms of allergy or asthma.
Heparin can be sprayed into the lungs of asthma sufferers and reduce symptoms. This suggests that the ratio of heparin to cytokines is important and that cytokine signaling required for asthma episodes of airway constriction can bind individually to different heparin molecules and minimize mast cell triggering and histamine release.

Asthma also responds to a general decrease in chronic systemic inflammation. Thus, an anti-inflammatory diet and lifestyle, can reduce episodes and potentially reverse symptoms. Omega-3 oils and glucosamine, for example are both effective.

Tryptase model: Sommerhoff CP, Bode W, Pereira PJ, Stubbs MT, Stürzebecher J, Piechottka GP, Matschiner G, Bergner A. 1999. The structure of the human betaII-tryptase tetramer: fo(u)r better or worse. Proc Natl Acad Sci U S A 96(20):10984-91.


Berberine staining of mast cell heparin: Feyerabend TB, Hausser H, Tietz A, Blum C, Hellman L, Straus AH, Takahashi HK, Morgan ES, Dvorak AM, Fehling HJ, Rodewald HR. 2005. Loss of histochemical identity in mast cells lacking carboxypeptidase A. Mol Cell Biol. 25:6199-210.