Low-level laser treatment accelerated hair regrowth in a rat model of chemotherapy-induced alopecia (CIA).

Chemotherapy-induced alopecia (CIA) is one of the most distressing side effects of antineoplastic chemotherapy for which there is no effective interventional approach. A low-level laser (LLL) device, the HairMax LaserComb®, has been cleared by the FDA to treat androgenetic alopecia. Its effects may be extended to other settings; we have demonstrated that LaserComb treatment induced hair regrowth in a mouse model for alopecia areata. In the current study, we tested whether LLL treatment could promote hair regrowth in a rat model for CIA. Chemotherapy agents cyclophosphamide, etoposide, or a combination of cyclophosphamide and doxorubicin were administered in young rats to induce alopecia, with or without LLL treatment. As expected, 7-10 days later, all the rats developed full body alopecia. However, rats receiving laser treatment regrew hair 5 days earlier than rats receiving chemotherapy alone or sham laser treatment (with the laser turned off). The accelerated hair regrowth in laser-treated rats was confirmed by histology. In addition, LLL treatment did not provide local protection to subcutaneously injected Shay chloroleukemic cells. Taken together, our results demonstrated that LLL treatment significantly accelerated hair regrowth after CIA without compromising the efficacy of chemotherapy in our rat model. Our results suggest that LLL should be explored for the treatment of CIA in clinical trials because LLL devices for home use (such as the HairMax LaserComb®) provide a user-friendly and noninvasive approach that could be translated to increased patient compliance and improved efficacy.

Effects of the Lexington LaserComb on hair regrowth in the C3H/HeJ mouse model of alopecia areata.

Alopecia areata (AA) is a common autoimmune disease that presents with non-scarring alopecia. It is characterized by intra- or peri-follicular lymphocytic infiltrates composed of CD4+ and CD8+ T-cells on histology. To this day, few treatments are effective for AA. Here we present findings of using a low-level laser comb to alleviate the symptoms of AA in a C3H/HeJ mouse model for AA. Fourteen C3H/HeJ mice with induced AA were used in this study. Two were killed to confirm AA through histology. The remaining 12 mice were randomized into two groups; group I received HairMax LaserComb (wavelength: 655 nm, beam diameter <5 mm; divergence 57 mrad; nine lasers) for 20 s daily, three times per week for a total of 6 weeks; group II was treated similarly, except that the laser was turned off (sham-treated). After 6 weeks of LaserComb treatment, hair regrowth was observed in all the mice in group I (laser-treated) but none in group II (sham-treated). On histology, increased number of anagen hair follicles was observed in laser-treated mice. On the other hand, sham-treated mice demonstrated hair follicles in the telogen phase with no hair shaft. LaserComb seems to be an effective and convenient device for the treatment of AA in the C3H/HeJ mouse model. Human studies are required to determine the efficacy and safety of this device for AA therapy.

Prevention and treatment of alopecia areata with quercetin in the C3H/HeJ mouse model.

Alopecia areata (AA) is an autoimmune non-scarring hair loss disorder. AA can be acute, recurrent, or chronic. Current therapeutic options for AA are limited, and there is no effective prevention for recurrent AA. We have previously shown a correlation between the expression of HSP70 (HSPA1A/B), a heat shock protein involved in the inflammatory response, and the onset of AA in the C3H/HeJ mouse model. In this study, we tested the effects of quercetin, a bioflavonoid with anti-inflammatory properties, on AA development and HSP70 expression in the C3H/HeJ model. Mice with spontaneous AA were treated with subcutaneous quercetin or sham injections. Hair regrowth was observed in lesional areas in all the quercetin-treated mice, but in none of the sham-treated mice. In addition, non-alopecic C3H/HeJ mice were heat-treated to induce alopecia, along with quercetin or sham injections. Whereas 24% of the heat-treated mice with sham injections developed alopecia, none of the mice receiving quercetin injections did. As expected, the level of HSP70 expression in quercetin-treated areas was comparable to control. Furthermore, we showed that systemic delivery of quercetin by intraperitoneal injections prevented/reduced spontaneous onset of AA. Our results demonstrated that quercetin provided effective treatment for AA as well as prevention of onset of AA in the C3H/HeJ model, and warrant further clinical studies to determine whether quercetin may provide both treatment for preexisting AA and prevention of recurrent AA. The ready availability of quercetin as a dietary supplement may lead to increased patient compliance and positive outcomes for AA.

Cross-section Trichometry: A Clinical Tool for Assessing the Progression and Treatment Response of Alopecia.

BACKGROUND: To properly assess the progression and treatment response of alopecia, one must measure the changes in hair mass, which is influenced by both the density and diameter of hair. Unfortunately, a convenient device for hair mass evaluation had not been available to dermatologists until the recent introduction of the cross-section trichometer, which directly measures the cross-sectional area of an isolated bundle of hair. OBJECTIVE: We sought to evaluate the accuracy and sensitivity of the HairCheck(®) device, a commercial product derived from the original cross-section trichometer. MATERIALS AND METHODS: Bundles of surgical silk and human hair were used to evaluate the ability of the HairCheck(®) device to detect and measure small changes in the number and diameter of strands, and bundle weight. RESULTS: Strong correlations were observed between the bundle's cross-sectional area, displayed as the numeric Hair Mass Index (HMI), the number of strands, the silk/hair diameter, and the bundle dry weight. CONCLUSION: HMI strongly correlated with the number and diameter of silk/hair, and the weight of the bundle, suggesting that it can serve as a valid indicator of hair mass. We have given the name cross-section trichometry (CST) to the methodology of obtaining the HMI using the HairCheck(®) system. CST is a simple modality for the quantification of hair mass, and may be used as a convenient and useful tool to clinically assess changes in hair mass caused by thinning, shedding, breakage, or growth in males and females with progressive alopecia or those receiving alopecia treatment.

Heat treatment increases the incidence of alopecia areata in the C3H/HeJ mouse model.

Alopecia areata (AA) is a common autoimmune disease characterized by non-scarring hair loss. Previous studies have demonstrated an association between AA and physiological/psychological stress. In this study, we investigated the effects of heat treatment, a physiological stress, on AA development in C3H/HeJ mice. Whereas this strain of mice are predisposed to AA at low incidence by 18 months of age, we observed a significant increase in the incidence of hair loss in heat-treated 8-month-old C3H/HeJ mice compared with sham-treated mice. Histological analysis detected mononuclear cell infiltration in anagen hair follicles, a characteristic of AA, in heat-treated mouse skin. As expected, increased expression of induced HSPA1A/B (formerly called HSP70i) was detected in skin samples from heat-treated mice. Importantly, increased HSPA1A/B expression was also detected in skin samples from C3H/HeJ mice that developed AA spontaneously. Our results suggest that induction of HSPA1A/B may precipitate the development of AA in C3H/HeJ mice. For future studies, the C3H/HeJ mice with heat treatment may prove a useful model to investigate stress response in AA.

Prevention of chemotherapy-induced alopecia in rodent models.

Alopecia (hair loss) is experienced by thousands of cancer patients every year. Substantial-to-severe alopecia is induced by anthracyclines (e.g., adriamycin), taxanes (e.g., taxol), alkylating compounds (e.g., cyclophosphamide), and the topisomerase inhibitor etoposide, agents that are widely used in the treatment of leukemias and breast, lung, ovarian, and bladder cancers. Currently, no treatment appears to be generally effective in reliably preventing this secondary effect of chemotherapy. We observed in experiments using different rodent models that localized administration of heat or subcutaneous/intradermal injection of geldanamycin or 17-(allylamino)-17-demethoxygeldanamycin induced a stress protein response in hair follicles and effectively prevented alopecia from adriamycin, cyclophosphamide, taxol, and etoposide. Model tumor therapy experiments support the presumption that such localized hair-saving treatment does not negatively affect chemotherapy efficacy.

Transendothelial migration of leukocytes is promoted by plasma from a subgroup of immune thrombocytopenic purpura patients with small-vessel ischemic brain disease.

We previously described a subgroup of immune thrombocytopenic purpura (ITP) patients presenting with recurring transient ischemic attack-like symptoms and progressive cognitive impairment due to small vessel disease (SVD) seen in the brain. They presented minimal bleeding despite thrombocytopenia, and platelet activation was elevated compared to classic ITP. On the hypothesis that the blood-brain barrier (BBB) is compromised in this subgroup, we investigated the effect of plasma from SVD-ITP patients on the transendothelial migration of leukocytes (TEML). Brain microvascular endothelial cells (BMVEC) were grown to confluence on 6.5-microm pore filters and plasma from 10 healthy controls, 20 classic ITP, and 5 SVD-ITP were added and incubated 24 hr. Then 1 x 10(5) monocytes (U937) were added and the number migrated through the EC monolayer after 6 hr was measured by flow cytometry. The effect on TEML of danazol was also assessed. We found that plasma from SVD-ITP but not classic ITP induced 10-fold rise in EC activation marker CD62E and a sevenfold increase in TEML, to 38.5% +/- 12.5% of cells migrated, compared to normal controls (5.6% +/- 1.2%) or classic ITP (6.1% +/- 0.2%), P < 0.001. Preincubation of U937 with endothelial microparticles (EMP) increased TEML by 20.0% +/- 6.4% with SVD-ITP plasma, significantly more than with classic ITP or control plasmas, P = 0.003. Pretreatment of cultures with danazol (100 microg/mL) inhibited TEML by 25% in all wells tested, whether or not EMP were added. In summary, SVD-ITP plasma activates EC and augments TEML, suggesting plasma-mediated BBB dysfunction in this syndrome. Danazol modestly but significantly inhibited TEML.

Elevated endothelial microparticle-monocyte complexes induced by multiple sclerosis plasma and the inhibitory effects of interferon-beta 1b on release of endothelial microparticles, formation and transendothelial migration of monocyte-endothelial microparticle complexes.

Monocyte migration through the disrupted cerebral endothelial cell (EC) junctions plays an essential role in formation of multiple sclerosis (MS) demyelinating lesions. During pathogenesis of MS, activated ECs release endothelial microparticles (EMP), which possibly facilitate transendothelial migration (TEMIG) of monocytes. To assess functional roles of EMP in MS, specifically, their (i) interaction with monocytes, (ii) effect on monocyte TEMIG in an in vitro model of the brain microvascular endothelial cells (BMVEC), (iii) phenotypic profiles of EMP elicited by MS plasma and (iv) the effects of IFN-beta 1b on release of EMP and on TEMIG of monocytes (mono) and monocytes:EMP complexes (mono:EMP) through the BMVEC. The effect of IFN-beta 1b on the release of EMP and the TEMIG of mono and mono:EMP was assessed by preincubating BMVEC cultures of IFN-beta 1b prior to addition of plasma. Three EMP phenotypes, CD54, CD62E and CD31 were assayed. Plasma specimens from 20 patients with relapsing remitting MS (11 in exacerbation, MS-E, and 9 in remission, ME-R) and 10 healthy controls were studied. Incubation of BMVEC with MS-E plasma yielded elevated levels of EMPCD54, EMP62E and EMPCD31 relative to MS-R and control plasmas. MS-E but not MS-R or control plasma also augmented TEMIG of monocytes, respectively. Mono:EMP complexes further augmented TEMIG relative to mono alone, but only in the presence of MS-E plasma; there was no significant effect with MS-R or control plasmas. The presence of IFN-beta 1b inhibited TEMIG of mono and mono:EMP by 20% and 30%, respectively. MS-E but not MS-R plasma elicited release of activation-derived EMP and enhanced TEMIG of mono and mono:EMP. IFN-beta 1b inhibited TEMIG and release of EMP, suggesting a role of EMP and a novel therapeutic mechanism for IFN-beta 1b in MS.

Postprandial hypertriglyceridemia increases circulating levels of endothelial cell microparticles.

BACKGROUND: This study evaluated a possible relationship between levels of endothelial microparticles (EMPs), known to be a sensitive indicator of endothelial disturbance, and changes in postprandial lipid levels in healthy volunteers after a low- or high-fat meal. METHODS AND RESULTS: Eighteen healthy subjects without known cardiovascular risk factors were evaluated. Lipid and EMP levels were measured before and 1 and 3 hours after a single low- or high-fat isocaloric meal. The low-fat meal had no significant postprandial effect on EMPs or lipids compared with fasting levels. In contrast, a single high-fat meal significantly increased EMP levels after 1 and 3 hours, from 389+/-54 (thousands per milliliter) when fasting to 541+/-139 (P=0.0002) and 677+/-159 (P<0.0001), respectively, and correlated with a postprandial elevation in serum triglycerides. CONCLUSIONS: A single high-fat meal led to a significant elevation of plasma EMP levels in healthy, normolipidemic subjects and correlated with a postprandial elevation of serum triglycerides. EMPs may be an indirect marker of endothelial dysfunction or injury induced by postprandial triglyceride-rich lipoproteins.

Elevated plasma endothelial microparticles: preeclampsia versus gestational hypertension.

OBJECTIVE: Elevated plasma endothelial microparticle levels have been found to be elevated in women with preeclampsia. However, their role in distinguishing preeclampsia from gestational hypertension remains to be elucidated. The objectives of this study were to compare endothelial microparticle levels among patients with preeclampsia, gestational hypertension, and healthy pregnant control subjects and to evaluate the effect of plasma from women with preeclampsia and gestational hypertension on the release of endothelial microparticles by renal microvascular endothelial cells. STUDY DESIGN: A prospective study was conducted on 52 women with preeclampsia, 20 women with gestational hypertension, and 38 healthy pregnant control subjects. Endothelial microparticles were measured by flow cytometry with fluorescent monoclonal mouse anti-human antibodies against CD31, CD42b, and CD62E. RESULTS: CD31 + /42b - endothelial microparticle levels were 10497 +/- 5145 counts/microL in women with preeclampsia versus 6768 +/- 1810 counts/microL in women with gestational hypertension ( P < .01). In control subjects, CD31 + /42b - endothelial microparticle levels were 6119 +/- 3592 counts/microL. CD62E + endothelial microparticle levels were 1930 +/- 966 counts/microL in women with preeclampsia versus 822 +/- 150 counts/microL in women with gestational hypertension ( P <.01). In control subjects, CD62E + endothelial microparticle levels were 712 +/- 160 counts/microL. Incubation of renal microvascular endothelial cells with plasma from women with preeclampsia resulted in a rise in CD31 + and CD62E + endothelial microparticle levels as compared with women with gestational hypertension and control subjects. CONCLUSION: Endothelial microparticle levels are higher in women with preeclampsia than in women with gestational hypertension and control subjects. The measurement of endothelial microparticles may be useful as a diagnostic tool for preeclampsia in pregnant women.

Endothelial microparticles (EMP) bind and activate monocytes: elevated EMP-monocyte conjugates in multiple sclerosis.

Elevated plasma endothelial microparticles (EMP) have been documented in MS during exacerbation. However, the role of EMP in pathogenesis of MS remains unclear. We investigated the formation of EMP-monocyte conjugates (EMP-MoC) and their potential role in transendothelial migration of inflammatory cells in MS. EMP-MoC were assayed in 30 MS patients in exacerbation, 20 in remission and in 35 controls. EMP-leukocyte conjugation was investigated flowcytometrically by employing alpha-CD54 or alpha-CD62E for EMP, and alpha-CD45 for leukocytes. EMP-MoC were characterized by identifying adhesion molecules involved and their effect on monocyte function. In vivo (clinical): EMP-MoC were markedly elevated in exacerbation vs. remission and controls, correlating with presence of GD+ MRI lesions. Free CD54+ EMP were not elevated but free CD62E+ EMP were. In vitro: EMP bound preferentially to monocytes, less to neutrophils, but little to lymphocytes. Bound EMP activated monocytes: CD11b expression increased 50% and migration through cerebral endothelial cell layer increased 2.6-fold. Blockade of CD54 reduced binding by 80%. Most CD54+ EMP bound to monocytes, leaving little free EMP, while CD62+ EMP were found both free and bound. These results demonstrated that phenotypic subsets of EMP interacted differently with monocytes. Based on our observations, EMP may enhance inflammation and increase transendothelial migration of monocytes in MS by binding to and activating monocytes through CD54. EMP-MoC were markedly increased in MS patients in exacerbation compared to remission and may serve as a sensitive marker of MS disease activity.

Endothelial microparticles released in thrombotic thrombocytopenic purpura express von Willebrand factor and markers of endothelial activation.

It has been suggested that endothelial apoptosis is a primary lesion in the pathogenesis of thrombotic thrombocytopenic purpura (TTP). We tested this hypothesis by examining the phenotypic signatures of endothelial microparticles (EMP) in TTP patients. In addition, the effect of TTP plasma on microvascular endothelial cells (MVEC) in culture was further delineated. EMP released by endothelial cells (EC) express markers of the parent EC; EMP released in activation carry predominantly CD54 and CD62E, while those in apoptosis CD31 and CD105. We investigated EMP release in vitro and in TTP patients. Following incubation of MVEC with TTP plasma, EMP and EC were analysed by flow cytometry for the expression of CD31, CD51, CD54, CD62E, CD105, CD106 and von Willebrand factor (VWF) antigen. EMP were also analysed in 12 TTP patients. In both EC and EMP, CD62E and CD54 expression were increased 3- to 10-fold and 8- to 10-fold respectively. However, CD31 and CD105 were reduced 40-60% in EC but increased twofold in EMP. VWF expression was found in 55 +/- 15% of CD62E+ EMP. Markers of apoptosis were negative. In TTP patients, CD62E+ and CD31+/CD42b- EMP were markedly elevated, and preceded and correlated well with a rise in platelet counts and a fall in lactate dehydrogenase. CD62E+ EMP (60 +/- 20%) co-expressed VWF and CD62E. The ratio of CD31+/42b- to CD62E+ EMP exhibited a pattern consistent with activation. In conclusion, our studies indicate endothelial activation in TTP. EMP that co-express VWF and CD62E could play a role in the pathogenesis of TTP.

High levels of circulating endothelial microparticles in patients with acute coronary syndromes.

BACKGROUND: Endothelial injury plays a critical role in coronary artery disease (CAD), but the assessment of this injury has been problematical. Recently, it has been shown in vitro that endothelial cells (ECs) release endothelial microparticles (EMPs) on activation or apoptosis and that an assay of EMPs can provide useful information on EC status in patients with thrombotic disorders. This study is aimed at assessing possible correlations between EMPs, which are markers of endothelial injury, and clinical subgroups of patients with CAD. METHODS: A prospective, case-controlled study was conducted on 84 patients with CAD and 42 control subjects to investigate EMP profiles. Included were 64 patients with acute coronary syndromes ([ACS], 38 with myocardial infarction [MI] and 26 with unstable angina [UA]) and 20 patients with stable angina (SA). EMPs in platelet-poor plasma were measured flow cytometrically with combinations of fluorescent antibodies (anti-CD31, -51, -42), allowing distinction of EMPs from platelet microparticles (PMPs). Clinical subgroups of patients were correlated with EMP and PMP levels in blood. RESULTS: Two species of EMPs (CD31+ and CD51+) were evaluated. Both were significantly higher in patients with CAD than in control subjects. CD31+ EMP was higher in ACS than SA. Among patients with first MI, CD31+ EMP was higher in patients with MI than in patients with UA and was significantly higher than in patients with recurring MI. CD51+ EMP did not discriminate ACS from SA. A simultaneous assay of PMP showed correlation between EMPs and PMPs. However, PMPs did not discriminate patients with SA from control subjects. CONCLUSIONS: EMP assay appears promising for assessing EC injury in CAD.

Endothelial cells release phenotypically and quantitatively distinct microparticles in activation and apoptosis.

BACKGROUND: Endothelial cells (EC) shed endothelial microparticles (EMP) in activation and apoptosis. OBJECTIVES: We compared the antigenic expression of EMP species released during activation as compared to apoptosis, in three cell lines. METHODS: EC from renal and brain microvascular (MiVEC) and coronary macrovascular (MaVEC) origin were incubated with TNF-alpha to induce activation, or deprived of growth factors to induce apoptosis. Antigens expressed on EMP and EC were assayed flow cytometrically and included constitutive markers (CD31, CD51/61, CD105), inducible markers (CD54, CD62E and CD106), and annexin V binding. RESULTS: It was found that in apoptosis, constitutive markers in EMP were markedly increased (CD31>CD105), with a concomitant decrease in expression in EC. Annexin V EC surface binding and annexin V+ EMP were more sharply increased in apoptosis than in activation. In contrast, in activation, inducible markers in EMP were markedly increased in both EMP and EC (CD62E>CD54>CD106). Coronary MaVEC released significantly less EMP than MiVEC. CONCLUSION: EC release qualitatively and quantitatively distinct EMP during activation compared to apoptosis. Analysis of EMP phenotypic signatures may provide clinically useful information on the status of the endothelium.

Effects of severe hypertension on endothelial and platelet microparticles.

The molecular mechanisms by which extreme blood pressure elevation leads to vascular injury are not defined. To explore the hypothesis that activation of endothelium and platelets as manifested by increased concentrations of circulating endothelial microparticles and platelet microparticles could play a role in this target organ injury, we conducted a cross-sectional study of these markers in 3 groups: (1) untreated patients referred specifically for treatment of severe uncontrolled hypertension; (2) untreated patients with established mild hypertension; and (3) normotensive volunteer subjects. By ANOVA, endothelial (P=0.002) and platelet (P=0.01) microparticles were greatest in the severely hypertensive group. There was a significant correlation between both of these markers and blood pressure, even in the setting of multiple risk factors. Our results suggest that these markers may be useful and specific for pressure-induced endothelial and platelet activation in hypertension. Furthermore, because of the combined effects of endothelial and platelet microparticles on coagulation, leukocytes, and endothelium, it is possible that they may play a pathogenic role in mediating target organ injury in severe hypertension.

Agonist-induced capping of adhesion proteins and microparticle shedding in cultures of human renal microvascular endothelial cells.

Capping and release of membranous, small (< 1.5 microm) endothelial microparticles were quantified by immunofluorescence microscopy and flow cytometry after treatment of cultures of human renal microvascular endothelial cells with agonists tumor necrosis factor-alpha (TNF-alpha) or mitomycin C. For constitutive marker CD31, both agonist-treated attached, monolayer, and detached, free endothelial cells formed caps and released microparticles. TNF-alpha and mitomycin C induced dissimilar appearing CD31-containing caps after 3 h, followed by endothelial microparticle release after 6 h. The degree of capping correlated with increasing counts of released microparticles. For lymphokine-inducible CD54, TNF-alpha also induced CD54-containing caps and microparticle release, but mitomycin C failed to induce the expression of either entity. Neither capping nor microparticle release caused by TNF-alpha was part of an apoptotic pathway that involved caspase 3. Mitomycin C treatment of endothelial cells caused capping and microparticle release with a time course similar to TNF-alpha induction for 15 to 24 h, but assays for caspase 3 were positive, confirming the apoptotic action of mitomycin C. Membrane capping and microparticle release from endothelial cells are a convenient experimental model for studying protein movement, release of microparticles, and their possible biological significance.